Miniaturized cell array methods and apparatus for cell-based screening

ABSTRACT

The present invention describes methods and cassettes for cell-based toxin detection and organ localization. The cassettes includes an array containing cells and a matrix of openings or depressions, wherein each region of the substrate enclosed by the opening or depression in the matrix forms a domain individually addressable by microfluidic channels in the device.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. Nos. 60/145,757 filed Jul. 27, 1999; is a continuation of U.S. Pat.No. 7,160,687, filed Jul. 21, 2000, which is a continuation-in-part ofU.S. Pat. No. 6,548,263, filed Mar. 31, 2000, which is a continuation inpart of U.S. patent application Ser. No. 09/401,212 filed Sep. 22, 1999;which is a continuation in part of U.S. Pat. No. 6,103,479 filed May 29,1997; and is a continuation in part of U.S. Pat. No. 6,416,959 filedFeb. 25, 2000.

U.S. GOVERNMENT RIGHTS

This invention was made in part with support from the U.S. Governmentunder Contract No. N00014-98-C-0326, awarded by the U.S. Office of NavalResearch, an organization of the U.S. Department of Defense. The U.S.Government may have certain nonexclusive rights in this invention.

FIELD OF THE INVENTION

The present invention relates to methods and devices for cell-based highthroughput and high biological content screening.

BACKGROUND OF THE INVENTION

In the expanding arena of drug discovery and combinatorial chemistry togenerate candidate compounds, it would be very useful to be able torapidly screen a large number of substances, via a high throughputscreen, for their physiological impact on animals and humans. Beforetesting the efficacy of a “partially qualified” drug candidate onanimals, the drug could first be screened for its biological activityand potential toxicity with living cells. The physiological response tothe drug candidate could then be anticipated from the results of thesecell screens.

Traditionally, “lead compounds” have moved quickly to extensive animalstudies that are both time-consuming and costly. Furthermore, extensivedrug testing in animals is becoming less culturally acceptable.Screening drug candidates according to their interaction with livingcells, prior to animal studies, can reduce the number of animalsrequired in subsequent drug screening processes by eliminating some drugcandidates before going to animal trials. However, manipulation andanalysis of drug-cell interactions using current methods does not allowfor both high throughput and high biological content screening, due tothe small number of cells and compounds that can be analyzed in a givenperiod of time, the cumbersome methods required for compound delivery,and the large volumes of compounds required for testing.

High throughput screening of nucleic acids and polypeptides has beenachieved using DNA chip technologies. In typical DNA analysis methods,DNA sequences of 10 to 14 nucleotides are attached in defined locations(or spots), up to tens of thousands in number, on a small glass plate.(U.S. Pat. No. 5,556,752, hereby incorporated by reference). Thiscreates an array of spots of DNA on a given glass plate. The location ofa spot in the array provides an address for later reference to each spotof DNA. The DNA sequences are then hybridized with complementary DNAsequences labeled with fluorescent molecules. Signals from each addresson the array are detected when the fluorescent molecules attached to thehybridizing nucleic acid sequences fluoresce in the presence of light.These devices have been used to provide high throughput screening of DNAsequences in drug discovery efforts and in the human genome sequencingproject. Similarly, protein sequences of varying amino acid lengths havebeen attached in discrete spots as an array on a glass plate. (U.S. Pat.No. 5,143,854, incorporated by reference herein).

The information provided by an array of either nucleic acids or aminoacids bound to glass plates is limited according to their underlying“languages”. For example, DNA sequences have a language of only fournucleic acids and proteins have a language of about 20 amino acids. Incontrast, a living cell, which comprises a complex organization ofbiological components, has a vast “language” with a concomitantmultitude of potential interactions with a variety of substances, suchas DNA, RNA, cell surface proteins, intracellular proteins and the like.Because a typical target for drug action is with and within the cells ofthe body, cells themselves provide an extremely useful screening tool indrug discovery when combined with sensitive detection reagents. It thuswould be most useful to have high throughput, high content screeningdevices to provide high content spatial information at the cellular andsubcellular level as well as temporal information about changes inphysiological, biochemical and molecular activities.

Microarrays of Cells

Methods have been described for making micro-arrays of a single celltype on a common substrate for other applications. One example of suchmethods is photochemical resist-photolithograpy (Mrksich and Whitesides,Ann. Rev. Biophys. Biomol. Struct. 25:55-78, 1996), in which a glassplate is uniformly coated with a photoresist and a photo mask is placedover the photoresist coating to define the “array” or pattern desired.Upon exposure to light, the photoresist in the unmasked areas isremoved. The entire photolithographically defined surface is uniformlycoated with a hydrophobic substance, such as an organosilane, that bindsboth to the areas of exposed glass and the areas covered with thephotoresist. The photoresist is then stripped from the glass surface,exposing an array of spots of exposed glass. The glass plate then iswashed with an organosilane having terminal hydrophilic groups orchemically reactable groups such as amino groups. The hydrophilicorganosilane binds to the spots of exposed glass with the resultingglass plate having an array of hydrophilic or reactable spots (locatedin the areas of the original photoresist) across a hydrophobic surface.The array of spots of hydrophilic groups provides a substrate fornon-specific and non-covalent binding of certain cells, including thoseof neuronal origin (Kleinfeld et al., J. Neurosci. 8:4098-4120, 1988).

In another method based on specific yet non-covalent interactions,stamping is used to produce a gold surface coated with proteinadsorptive alkanethiol. (U.S. Pat. No. 5,776,748; Singhvi et al.,Science 264:696-698, 1994). The bare gold surface is then coated withpolyethylene-glycol-terminated alkanethiols that resist proteinadsorption. After exposure of the entire surface to laminin, acell-binding protein found in the extracellular matrix, livinghepatocytes attach uniformly to, and grow upon, the laminin coatedislands (Singhvi et al. 1994). An elaboration involving strong, butnon-covalent, metal chelation has been used to coat gold surfaces withpatterns of specific proteins (Sigal et al., Anal. Chem. 68:490-497,1996). In this case, the gold surface is patterned with alkanethiolsterminated with nitriloacetic acid. Bare regions of gold are coated withtri(ethyleneglycol) to reduce protein adsorption. After adding Ni²⁺, thespecific adsorption of five histidine-tagged proteins is found to bekinetically stable.

More specific single cell-type binding can be achieved by chemicallycrosslinking specific molecules, such as proteins, to reactable sites onthe patterned substrate. (Aplin and Hughes, Analyt. Biochem.113:144-148, 1981). Another elaboration of substrate patterningoptically creates an array of reactable spots. A glass plate is washedwith an organosilane that chemisorbs to the glass to coat the glass. Theorganosilane coating is irradiated by deep UV light through an opticalmask that defines a pattern of an array. The irradiation cleaves theSi—C bond to form a reactive Si radical. Reaction with water causes theSi radicals to form polar silanol groups. The polar silanol groupsconstitute spots on the array and are further modified to couple otherreactable molecules to the spots, as disclosed in U.S. Pat. No.5,324,591, incorporated by reference herein. For example, a silanecontaining a biologically functional group such as a free amino moietycan be reacted with the silanol groups. The free amino groups can thenbe used as sites of covalent attachment for biomolecules such asproteins, nucleic acids, carbohydrates, and lipids. The non-patternedcovalent attachment of a lectin, known to interact with the surface ofcells, to a glass substrate through reactive amino groups has beendemonstrated (Aplin & Hughes, 1981). The optical method of forming amicro-array of a single cell type on a support requires fewer steps andis faster than the photoresist method, (i.e., only two steps), but itrequires the use of high intensity ultraviolet light from an expensivelight source.

In all of these methods, the result is a micro-array of a single celltype, since the biochemically specific molecules are bound to themicro-patterned chemical array uniformly. In the photoresist method,cells bind to the array of hydrophilic spots and/or specific moleculesattached to the spots which, in turn, bind cells. Thus cells bind to allspots in the array in the same manner. In the optical method, cells bindto the array of spots of free amino groups by adhesion. There is littleor no differentiation between the spots of free amino groups. Again,cells adhere to all spots in the same manner, and thus only a singletype of cell interaction can be studied with these cell arrays becauseeach spot on the array is essentially the same as another. Such cellarrays are inflexible in their utility as tools for studying a specificvariety of cells in a single sample or a specific variety of cellinteractions. Thus, there exists a need for arrays of multiple celltypes on a common substrate, in order to increase the number of celltypes and specific cell interactions that can be analyzedsimultaneously, as well as methods of producing these micro-arrays ofmultiple cell types on a common substrate, in order to provide for highthroughput and high biological content screening of cells.

Optical Reading of Cell Physiology

Performing a high throughput screen on many thousands of compoundsrequires parallel handling and processing of many compounds and assaycomponent reagents. Standard high throughput screens use homogeneousmixtures of compounds and biological reagents along with some indicatorcompound, loaded into arrays of wells in standard microplates with 96 or384 wells. (Kahl et al., J. Biomol. Scr. 2:33-40, 1997). The signalmeasured from each well, either fluorescence emission, optical density,or radioactivity, integrates the signal from all the material in thewell giving an overall population average of all the molecules in thewell. This type of assay is commonly referred to as a homogeneous assay.

U.S. Pat. No. 5,581,487 describes an imaging plate reader that uses aCCD detector (charge-coupled optical detector) to image the whole areaof a 96 well plate. The image is analyzed to calculate the totalfluorescence per well for homogeneous assays.

Schroeder and Neagle describe a system that uses low angle laserscanning illumination and a mask to selectively excite fluorescencewithin approximately 200 microns of the bottoms of the wells in standard96 well plates in order to reduce background when imaging cellmonolayers. (J. Biomol. Scr. 1:75-80, 1996). This system uses a CCDcamera to image the whole area of the plate bottom. Although this systemmeasures signals originating from a cell monolayer at the bottom of thewell, the signal measured is averaged over the area of the well and istherefore still considered a homogeneous measurement, since it is anaverage response of a population of cells. The image is analyzed tocalculate the total fluorescence per well for cell-based homogeneousassays.

Proffitt et. al. (Cytometry 24:204-213, 1996) describe a semi-automatedfluorescence digital imaging system for quantifying relative cellnumbers in situ, where the cells have been pretreated with fluoresceindiacetate (FDA). The system utilizes a variety of tissue culture plateformats, particularly 96-well microplates. The system consists of anepifluorescence inverted microscope with a motorized stage, videocamera, image intensifier, and a microcomputer with a PC-Visiondigitizer. Turbo Pascal software controls the stage and scans the platetaking multiple images per well. The software calculates totalfluorescence per well, provides for daily calibration, and configuresfor a variety of tissue culture plate formats. Thresholding of digitalimages and the use of reagents that fluoresce only when taken up byliving cells are used to reduce background fluorescence without removingexcess fluorescent reagent.

A variety of methods have been developed to image fluorescent cells witha microscope and extract information about the spatial distribution andtemporal changes occurring in these cells. A recent article describesmany of these methods and their applications (Taylor et al., Am.Scientist 80:322-335, 1992). These methods have been designed andoptimized for the preparation of small numbers of specimens for highspatial and temporal resolution imaging measurements of distribution,amount and biochemical environment of the fluorescent reporter moleculesin the cells.

Treating cells with dyes and fluorescent reagents, imaging the cells,and engineering the cells to produce a fluorescent reporter molecule,such as modified green fluorescent protein (GFP), are useful detectionmethods (Wang et al., In Methods in Cell Biology, New York, Alan R.Liss, 29:1-12, 1989). The green fluorescent protein (GFP) of thejellyfish Aequorea victoria has an excitation maximum at 395 nm, anemission maximum at 510 nm and does not require an exogenous factor.Uses of GFP for the study of gene expression and protein localizationare discussed in Chalfie et al., Science 263:802-805, 1994. Someproperties of wild-type GFP are disclosed by Morise et al. (Biochemistry13:2656-2662, 1974), and Ward et al. (Photochem. Photobiol. 31:611-615,1980). An article by Rizzuto et al. (Nature 358:325-327, 1992) discussesthe use of wild-type GFP as a tool for visualizing subcellularorganelles in cells. Kaether and Gerdes (FEBS Letters 369:267-271, 1995)report the visualization of protein transport along the secretorypathway using wild-type GFP. The expression of GFP in plant cells isdiscussed by Hu and Cheng (FEBS Letters 369:331-334, 1995), while GFPexpression in Drosophila embryos is described by Davis et al. (Dev.Biology 170:726-729, 1995). U.S. Pat. No. 5,491,084, incorporated byreference herein, discloses expression of GFP from Aequorea victoria incells as a reporter molecule fused to another protein of interest.Mutants of GFP have been prepared and used in several biologicalsystems. (Hasselhoff et al., Proc. Natl. Acad. Sci. 94:2122-2127, 1997;Brejc et al., Proc. Natl. Acad. Sci. 94:2306-2311, 1997; Cheng et al.,Nature Biotech. 14:606-609, 1996; Heim and Tsien, Curr. Biol. 6:178-192,1996; Ehrig et al., FEBS Letters 367:163-166, 1995).

The ARRAYSCAN™ System, as developed by Cellomics, Inc. (U.S. Pat. No.5,989,835) and U.S. application Ser. No. 09/031,271 filed Feb. 27, 1998;both incorporated by reference herein in their entirety) is an opticalsystem for determining the distribution, environment, or activity ofluminescently labeled reporter molecules on or in cells for the purposeof screening large numbers of compounds for specific biologicalactivity. The ARRAYSCAN™ System involves providing cells containingluminescent reporter molecules in an array of locations and scanningnumerous cells in each location, converting the optical information intodigital data, and utilizing the digital data to determine thedistribution, environment or activity of the luminescently labeledreporter molecules in the cells. The ARRAYSCAN™ System includesapparatus and computerized method for processing, displaying and storingthe data, thus augmenting drug discovery by providing high contentcell-based screening, as well as combined high throughput and highcontent cell-based screening, in a large microplate format.

Microfluidics

Efficient delivery of solutions to an array of cells attached to a solidsubstrate is facilitated by a microfluidic system. Methods and apparatushave been described for the precise handling of small liquid samples forink delivery (U.S. Pat. No. 5,233,369; U.S. Pat. No. 5,486,855; U.S.Pat. No. 5,502,467), biosample aspiration (U.S. Pat. No. 4,982,739),reagent storage and delivery (U.S. Pat. No. 5,031,797), and partitionedmicroelectronic and fluidic device array for clinical diagnostics andchemical synthesis (U.S. Pat. No. 5,585,069). In addition, methods andapparatus have been described for the formation of microchannels insolid substrates that can be used to direct small liquid samples alongthe surface (U.S. Pat. No. 5,571,410; U.S. Pat. No. 5,500,071; U.S. Pat.No. 4,344,816,).

For purposes of integrated high throughput and high content cell basedscreening, particularly for live-cell imaging, an optimal microfluidicdevice would comprise a fluidic architecture that permits the closestpossible well spacing (i.e.: highest possible well density), wherein thefluidic architecture is integrated with the cell array substrate topermit efficient fluid delivery to the cells, and eliminating the needfor pipetting fluids in and out of wells. Such optimal microfluidicdevices would be advantageous for cell arrays with sub-millimeterinter-well distances because it is unwieldy, if not impossible, topipette fluids with such a high degree of spatial resolution andaccuracy. Furthermore, such integrated devices could be directly usedfor cell based screening, without the need to remove the cell substratefrom the fluidic architecture for imaging the cells.

An optimal microfluidic device for cell based screening might furthercomprise a closed chamber to permit environmental control of the cells,and preferably would not directly expose the cells to electro-kineticforces, which may affect the physiology of the cells on the substrate.For example, electrohydrodynamic pumping is less effective with polarsolvents (Marc Madou, Fundamentals of Microfabrication, CRC Press, BocaRaton, 1997, p. 433). Electro-osmosis is typically accompanied by somedegree of electrophoretic separation of charged medium components, suchas proteins.

U.S. Pat. No. 5,603,351 (‘the 351 patent’) describes a microfluidicdevice that uses a multilevel design consisting of two upper levels withchannels and a bottom level with reaction wells. However, this device isnot designed for use in cell based screening. The '351 patent does notdisclose a substrate containing cells or cell binding sites. Thedisclosed microfluidic network is designed to allow two or more reagentsto be combined in a reaction well, as opposed to an optimal cellscreening microfluidic system that allows living cells cultured on thewell bottoms to be exposed in serial fashion to two or more differentfluids. The '351 patent discloses a device with the wells etched intothe substrate at a maximal well density of 50 wells/inch². Furthermore,the substrate must be detached from the fluidic array for incubationand/or analysis. Finally, the '351 patent discloses a system ofelectrically-controlled electrohydrodynamic valves within the matrix ofthe wells that are less effective with aqueous media used in cellculture, and also may limit the degree of close-spacing between wells inthe array of wells.

U.S. Pat. No. 5,655,560 discloses a clog-free valving system, comprisinga fluid distribution system with multiple inputs and multiple outputsincorporating a crossed array of microchannels connected vertically atcrossing points by teflon valves. However, this patent does not disclosea substrate containing a cell array, nor an integrated fluidic device incombination with the substrate, nor a well density that is optimal forcell-based screening.

U.S. Pat. No. 5,900,130 (the '130′ patent) describes the active,electronic control of fluid movement in an interconnected capillarystructure. This patent does not teach a fluidic architecture thatmaximizes the area of the cell substrate that can be occupied by cellbinding sites. Nor does this patent disclose a substrate containing acell array, nor an integrated fluidic device in combination with thesubstrate. Furthermore, the patent only teaches the control of fluidflow by application of an electric field to the device.

U.S. Pat. No. 5,910,287 describes multi-well plastic plates forfluorescence measurements of biological and biochemical samples,including cells, limited to plates with greater than 864 wells. Thispatent does not describe a microfluidic device with a fluidicarchitecture integrated with the cell array substrate. Nor does thepatent disclose a closed chamber to permit environmental control of thecells on the substrate.

Thus, none of these prior microfluidic devices provide a fluidicarchitecture that permits the closest possible well spacing (i.e.:highest possible well density), wherein the fluidic architecture isintegrated with the cell array substrate to permit efficient fluiddelivery to the cells, and thus eliminating the need for pipettingfluids in and out of wells. Furthermore, prior microfluidic devices thatcomprise an array of wells use electrically-controlledelectrohydrodynamic valves within the matrix of the wells that would beless effective if used with aqueous media for cell culture, and whichlimit the well density.

While the above advances in cell array, optical cell physiology reading,and microfluidic technologies provide supporting technologies that canbe applied to improved high throughput and high content cell-basedscreening, there remains a need in the art for integrated devices andmethods that further decrease the amount of time necessary for suchscreening, as well as for devices and methods that further improve theability to conduct high throughput and high content cell-based screeningand the ability to flexibly and rapidly switch from one to the other. Inparticular, devices and methods that maximize the well density, therebyincreasing the number of wells that can be imaged in at one time, andthus greatly increasing the throughput of a screen while maintainingadequate resolution of the image, would be very advantageous.

The drug discovery industry already uses 96- and 384-well microplatesand is in transition towards the use of 1536-well plates. However,further increases in well density using prior technology are unlikelybecause of the great difficulty of pipetting liquids in and out of verysmall diameter wells.

SUMMARY OF THE INVENTION

The present invention fulfills the need in the art for devices andmethods that decrease the amount of time necessary to conduct cell-basedscreening, and specifically combines the ability to conduct highthroughput and high content cell-based screening and to flexibly andrapidly switch from one to the other The invention provides devices andmethods for maximizing the number of wells that can be imaged at onetime while still obtaining adequate pixel resolution in the image. Thisresult has been achieved through the use of fluidic architectures thatmaximizes well density. The present invention thus provides aminiaturized microplate system with closed fluidic volumes that areinternally supplied with fluid exchange, and with wells that are closelyspaced to more rapidly detect spatially-resolved features of individualcells.

In one aspect, the present invention provides a cassette for cellscreening, comprising a substrate having a surface containing aplurality of cell binding locations; a fluid delivery system fordelivering reagents to the plurality of cell binding locations, whereinthe fluid delivery system comprises a multi-level chamber that mateswith the substrate, wherein the multi-level chamber comprises a crossedarray of microfluidic input channels and output channels and a pluralityof fluidic locations in fluid connection with the microfluidic inputchannels and output channels; and a plurality of wells, wherein anindividual well comprises the space defined by the mating of one cellbinding location and one fluidic location, and wherein the wells arepresent at a density of at least about 20 wells per square centimeter.

In another aspect, the present invention provides a cassette for cellscreening comprising

a. a substrate having a surface, wherein the surface contains aplurality of cell binding locations;

b. a fluid delivery system for delivering reagents to the plurality ofcell binding locations, wherein the fluid delivery system comprises amulti-level chamber that mates with the substrate, wherein themulti-level chamber comprises

-   -   i. a crossed array of microfluidic input channels and output        channels, wherein each well is in fluid connection with one or        more input channels and one or more output channels;    -   ii. a plurality of fluidic locations in fluid connection with        the microfluidic input channels and output channels;    -   iii. one or more input manifolds in fluid connection with the        microfluidic input channels;    -   iv. one or more output manifolds in fluid connection with the        microfluidic output channels;    -   v. at least one source receptacle in fluid connection with the        one or more input manifolds; and    -   vi. at least one waste receptacle in fluid connection with one        or more output manifolds; and

c. a plurality of wells, wherein an individual well comprises the spacedefined by the mating of one cell binding location and one fluidiclocation.

In preferred embodiments, both of the above cassettes further comprise apump to control fluid flow within the microfluidic device; a temperaturecontroller of the substrate and/or a controller to regulate oxygen andcarbon dioxide partial pressures.

In another aspect, the present invention provides an improved method fordiffusion control in a cassette, wherein the improvement comprisesconstantly applying a passive restoring force to valves located withinmicrofluidic channels of the cassette.

In a still further aspect, the present invention provides a method forcell screening, comprising

a) providing an array of locations which contain multiple cells;

b) providing an optical system to obtain images of the array oflocations;

c) serially imaging sub-arrays of the array of locations; and

d) acquiring data from each of the sub-arrays in parallel.

In a preferred embodiment, the array of locations is provided as acassette, such as those disclosed above.

In a further aspect, the present invention provides novel methods formaking a substrate for selective cell patterning, and the substratesthemselves, wherein the method comprises contacting reactive hydroxylgroups on the surface of a substrate with a hydroxyl-reactivebifunctional molecule to form a monolayer, and using stencils to depositcell repulsive or cell adhesive moieties to the substrate. In preferredembodiments the hydroxyl-reactive bifunctional molecule is anaminosilane and the cell repulsive moiety comprises tresyl-activatedpolyethylene glycol.

Among other uses, the devices and methods of the present invention areideal for high content and/or high throughput cell-based screening. Thedevice of the invention is also ideally suited as a cell support systemfor a hand-held diagnostic device (i.e.: a miniaturized imagingcell-based assay system). The smaller format and sealed containment ofthe present device enables its use in a rugged, portable system. Thereis a great economic advantage from the use of higher density plates.There is a further economic advantage in faster imaging of sub-arrayswhen using a high density plate; this advantage is not fully realized ifthe distances between adjacent wells are not minimized. The presentinvention provides both of these advantages.

The present invention further provides cassettes for cell screening,comprising a substrate having a surface; a fluid delivery system matedwith the substrate, wherein the fluid delivery system comprises:

-   -   1) a matrix of openings or depressions, wherein each region of        the substrate enclosed by the opening or depression in the        matrix comprises an individually addressable domain; and    -   2) microfluidic channels that supply fluid to the domains.

In preferred embodiments, each domain comprises cell binding sitesseparated by cell repulsive regions and each domain comprises a singlecell type expressing one or more biosensors, arrayed on the cell bindingsites. In a more preferred embodiment, at least three different celltypes are arrayed on the surface, and each of the three different celltypes are specific for different tissue types.

The present invention further provides automated methods for cell basedtoxin detection and organ localization comprising providing an array oflocations containing multiple cell types derived from different organtypes and each of the cell types comprising at least one luminescentreporter molecule; wherein the localization, distribution, structure, oractivity of the at least one luminescent reporter molecule is altered bya toxin to be detected; contacting the cells with a test substance andutilizing digital data derived from the reporter molecules to detect thepresence of a toxin in the compound, and to characterize itsbiodistribution.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a top view of a small substrate micro-patterned chemicalarray.

FIG. 1B is a top view of a large substrate micro-patterned chemicalarray.

FIG. 2 diagrams a method of producing a micro-patterned chemical arrayon a substrate.

FIG. 3A is a photograph showing fibroblastic cell growth on a surfacepatterned chip, attached to a micro-patterned chemical array and labeledwith two fluorescent probes.

FIG. 3B is a photograph showing fibroblastic cell growth in spottedpatterns, attached to a micro-patterned chemical array and labeled withtwo fluorescent probes.

FIG. 4 is a diagram of the cassette which is the combination of themicro-array of multiple cell types top and chamber bottom.

FIG. 5 is a diagram of a chamber that has nanofabricated input channelsto address “wells” in the non-uniform micro-patterned array of cells.

FIG. 6 is a diagram of a chamber with no channels.

FIG. 7A is an overhead diagram of a chamber with microfluidic channelsetched onto the substrate.

FIG. 7B is a side view diagram of a chamber with microfluidic channelsetched onto the substrate.

FIG. 8A is an overhead diagram of a chamber where the microfluidicchannels and wells are formed from a raised matrix of a material stampedonto the fluid delivery chamber.

FIG. 8B is a side view diagram of a chamber where the microfluidicchannels and wells are formed from a raised matrix of a material stampedonto the fluid delivery chamber.

FIG. 9 is a diagram of a chamber where each well is addressed by achannel originating from one side of the chamber.

FIG. 10 is a diagram of a chamber where the wells are addressed bychannels originating from two sides of the chamber.

FIG. 11 is a diagram of a chamber where the microfluidic switches arecontrolled by light, heat or other mechanical means.

FIG. 12 is a diagram of the luminescence reader instrument.

FIG. 13 is a diagram of one embodiment of the luminescence readerinstrument optical system.

FIG. 14A is a flow chart providing an overview of the cell screeningmethod.

FIG. 14B is a Macro (High Throughput Mode) Processing flow chart.

FIG. 14C is a Micro (High Content Mode) Processing flow chart.

FIG. 15 is a diagram of the integrated cell screening system.

FIG. 16 is a photograph of the user interface of the luminescence readerinstrument.

FIG. 17A is a photograph showing lymphoid cells nonspecifically attachedto an unmodified substrate.

FIG. 17B is a photograph showing lymphoid cells nonspecifically attachedto an IgM-coated substrate.

FIG. 17C is a photograph showing lymphoid cells specifically bound to awhole anti-serum-coated substrate.

FIG. 18A is a photographic image from High Throughput Mode ofluminescence reader instrument identifying “hits”.

FIG. 18B is a series of photographic images showing the high contentmode identifying high content biological information.

FIG. 19 is a photographic image showing the display of cell datagathered from the high content mode.

FIG. 20 depicts the optimization of cell imaging by simultaneous imagingof a sub-array of cell binding locations of the whole array. There is aone to one correspondence between the array of cell binding locations onthe substrate, the array of fluidic locations on the chamber, and thearray of wells formed by the joining of the two.

FIG. 21 exemplifies an 8×8 array (64 wells, 128 channels) of themicrofluidic device wherein each fluidic location is provided with aseparate input and output channel, yielding 2n² channels for an n×narray.

FIG. 22 depicts the microfluidic device of FIG. 21 wherein m+n² valvesor pumps are utilized to control the flow of m liquid or vapor mixturesto an 8×8 array.

FIG. 23 depicts an embodiment of the microfluidic device wherein thefluid delivery system consists of a crossed, nonintersecting array ofinput and output channels, and horizontal connecting channels utilizingmultiple levels, and wherein the two layers lie in the same plane of thewell layer.

FIG. 24 depicts an end view and side view of the embodiment of FIG. 23.

FIG. 25 depicts an embodiment of the microfluidic device wherein thefluid delivery system consists of a crossed, nonintersecting array ofinput and output channels, and vertical connecting channels utilizingmultiple levels, and wherein the two layers lie in a plane above that ofthe well layer.

FIG. 26 depicts an end view and side view of the embodiment of FIG. 25.

FIG. 27 shows an embodiment of the microfluidic device wherein compoundsare multiplexed to a single fluidic location by using m positivepressure source reservoirs or pumps, a valve-less manifold, andvalve-free waste reservoir at atmospheric pressure.

FIG. 28 shows an embodiment of the microfluidic device wherein compoundsare multiplexed to a single fluidic location by using m positivepressure source reservoirs or pumps, a valved manifold, or a negativepressure waste reservoir or pump with m valves connected to m sourcereservoirs at atmospheric pressure and valve-free waste reservoir atatmospheric pressure.

FIG. 29 shows an embodiment of the microfluidic device wherein the arrayof m positive pressure source reservoirs or pumps is multiplexed to ann×n crossed channel array of fluidic locations by means of 1×n input andn×1 output valve manifolds.

FIG. 30 shows an embodiment of the microfluidic device with m reservoirsat atmospheric pressure and a negative pressure waste reservoir by meansof 1×n input and n×1 output valve manifolds, where the negative pressurereservoir may be either mechanically or thermodynamically pumped.

FIG. 31 shows an embodiment a dual-pumped system, in which there is onlyone waste negative pressure reservoir that must be actuated incoordinated fashion with the actuation of any particular positivepressure source reservoir. This negative pressure reservoir may functionby either mechanical or thermodynamic (e.g., a capillary action pump)means. For the capillary action pump, the “actuation” of the pump isachieved by means of valves.

FIG. 32 shows how the pumping and valving scheme of FIG. 29, employingpositive pressure source reservoirs, can be extended to work with acrossed array of n row channels and 2n column channels (n ordinarycolumn channels plus n rinse column channels). This requires a 2n×1output valve manifold. Similarly, the scheme of FIG. 30, employing anegative pressure waste reservoir, can be extended to work with an arrayincorporating rinse channels.

FIG. 33 depicts an embodiment of the microfluidic device wherein everycolumn channel k has an associated parallel rinse channel k*.

FIG. 34 depicts an embodiment of the microfluidic device wherein thefluid delivery system consists of a crossed, nonintersecting array ofinput and output channels with vertical connecting channels, utilizingmultiple levels, wherein the two layers lie in a plane above that of thewell layer and every column channel k has an associated parallel rinsechannel k*.

FIG. 35 shows, in a case of fluid flow through well (j,k), an example ofa method to rinse a segment of liquid or vapor from row channel j andinto rinse channel k*, where k* is immediately adjacent to columnchannel k.

FIG. 36 depicts a method of diffusion control using “normally-closed”check valves, comprising a valve element and a valve seat, between everypair of adjacent vias that connect the channels to the fluidiclocations.

FIG. 37 depicts an embodiment wherein an externally-applied magneticfield gradient induces a restoring force on valve elements and valveseat that incorporate a magnetic material.

FIG. 38 shows an embodiment of a shaped valve seat wherein diffusionpast the ball is stopped when the externally-applied restoring forcepresses the ball to the left into a round opening in the valve seat.

FIG. 39 shows an embodiment that allows a single magnetic field gradientapplied to the whole microfluidic device to induce force on both a setof ball valves of the input channels and a set of ball valves of theoutput channels

FIG. 40 shows one embodiment of the pumping and valving schemes of thisinvention as it would appear with all the pumps and valves on-board thecassette.

FIG. 41 shows an embodiment, wherein the pumps are on-board and utilizeelectrically-driven flow, where the electric fields are limited toregions external to the matrix of the array of wells, and minimal or noelectric field gradients are applied across any of the wells in whichlive cells are present.

FIG. 42 (a) The standard structure of tresyl-PEG, wherein “n” can equalany number. (b) The structure oftrimethoxysilylpropyldiethylenetriamine.

FIG. 43 (a) An example of an amine-PEG surface product. (b) An exampleof a surface amine.

FIG. 44 is a most preferred embodiment for achieving selectivepositioning of cell adhesive molecules and cell repulsive moieties.

FIG. 45 is an experimental flowchart for selective differentiation ofstem cells on a substrate to produce both tissue-specific andorgan-specific cell substrates.

FIG. 46 shows the differential response in a dual labeling assay of thep38 MAPK and NF-κB pathways across three model toxins and two differentcell types. Treatments marked with an asterisk are different fromcontrols at a 99% confidence level (p<0.01).

FIG. 47 is a bar graph showing quantitative results for the proportionof NFKB protein in the nucleus versus the cytoplasm for wells treated bymedium exchange (negative control), TNF α (1.8 nM), Anisomycin (30 μM),and Cytochalasin D (2 μM) after exposure of Swiss 3T3 cells for 45minutes. The dominant response is obtained for TNF α. Mean+/−SEM n=1500cells totaled across 16 wells.

FIG. 48 is a bar graph showing quantitative results for the proportionof phosphorylated p38 MAPK in the nucleus versus the cytoplasm for wellstreated by medium exchange (negative control), TNF α (1.8 nM),Anisomycin (30 μM), and Cytochalasin D (2 μM) after exposure of Swiss3T3 cells for 45 minutes. The dominant response is obtained forAnisomycin. Mean+/−SEM n=1500 cells totaled across 16 wells.

FIG. 49 is a bar graph showing quantitative results for the proportionof phosphorylated p38 MAPK in the nucleus versus the cytoplasm for wellstreated by medium exchange (negative control), TNF α (1.8 nM),Anisomycin (30 μM), and Cytochalasin D (2 μM) after exposure of Swiss3T3 cells for 45 min. The dominant response is obtained for Anisomycin.Mean+/−SEM n=1500 cells totaled across 16 wells. TABLE 1 GUIDE TO THEFIGURES 001 fluidic location 002 array of fluidic locations, optionallywith depressions 003 sub-array 004 substrate 008 cell binding location009 chemical modification of the cell binding locations 010 cellsarrayed on the cell binding locations 012 chamber 013 depression formedat fluidic location 014 input channel 016 output channel 018 cassette020 spacer supports 024 micro-capillary tubes 028 raised reservoir 030first channel 032 second channel 036 plug 040 array of fluidic locations044 luminescence reader instrument 048 first storage compartment 050first robotic arm 052 second robotic arm 054 second storage compartment056 computer 060 Optics 064 computer controlled x-y-z stage 068computer-controlled rotating nosepiece 070 low-magnification objective072 high-magnification objective 074 while-light source lamp 076excitation filter wheel 078 dichroic filter system 080 emission filters082 detector (e.g., cooled CCD) 086 computer screen 090 database 140lateral space between wells 160 horizontal connecting channels 180vertical connecting channels 200 switchable positive pressure sourcereservoirs or pumps 220 source reservoirs at atmospheric pressure 240 m× 1 valve-less manifold 300 waste reservoirs 320 switchable negativepressure waste reservoirs or pumps 340 thermodynamically-pumped wastereservoir 400 1 × n valve manifold 410 n × 1 valve manifold 420 2n × 1valve manifold 500 Check Valve 520 Valve Element 540 Valve Seat 560magnetic (B) field gradient 580 restoring force on magnetic ball checkvalve 600 Bead 620 Magnetic Field 700 Fill ports 710 Channels 720 Valves730 Reservoirs 740 Electrokinetic pump 760 Electrode 1 780 Electrode 2

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

All patents, patent applications, and references cited herein areincorporated by reference in their entirety.

As used herein the term “well” is defined as the volume space created bythe mating of one fluidic location of the chamber with one cell bindinglocation on the substrate. The volume space may be created by adepression on the chamber corresponding to the fluidic location, by adepression on the substrate corresponding to the cell binding location,by a spacer support that separates the chamber and the substrate(wherein there is no requirement for a depression in the substrate ofthe chamber), a combination of any of these, or any other suitablemethod for creating a volume space between the fluidic location and thecell binding location.

As used herein, the term “cell binding location” refers to a discretelocation on the substrate that comprises a plurality of cell bindingsites capable of binding cells. The substrate may be derivatized tocreate cell binding sites, or the cell binding sites may naturally bepresent on the substrate. The plurality of cell binding sites within anindividual cell binding location may be capable of binding only a singlecell type, or may be capable of binding more than one cell type.Different cell binding locations on the same substrate may be identicalor different in the type of cells that they are capable of binding.

The term “fluidic location” as used herein refers to a discrete locationon the chamber that is the site of fluid delivery to and/or removal fromthe cell binding location. The fluidic location may comprise adepression, such as an etched domain, a raised reservoir, or any othertype of depression. Alternatively, the fluidic location may be flat,such as the terminus of an input and/or output channel, or the terminusof a via that is in fluid connection with an input and/or outputchannel.

As used herein, the term “cassette” means the combination of thesubstrate and the chamber. The combination can be either modular (morethan one piece), providing a re-usable chamber and a disposablesubstrate, or non-modular (single piece), to limit any potentialinterwell leaking.

As used herein, the term “on-board” means integral with the cassette.

As used herein, the term “integrated fluidics” means that a microfluidicdevice comprises both a substrate surface for cell binding and achamber.

As used herein, the term “chamber” means a fluid delivery systemcomprising microfluidic channels and fluidic locations, which serves asa specialized substrate cover.

As used herein, the term “switchable pump” refers to a pump, includingbut not limited to a syringe pump, a pressure-controlled vessel with avalve, or an electrokinetic pump that is employed externally (with theoptional use of electrical shielding) with respect to the matrix of thearray of wells, so that no harmful electric field effects areexperienced by the cells.

As used herein, the term “sub-array” means any contiguous sub-section ofwells in the entire array of wells, typically in a format correspondingto the shape of an imaging detector array, such as a CCD (charge coupleddevice detector).

As used herein the term “matrix of the cell array” means the spacedefined by the imaginary parallelpiped (i.e., typically a cubic orrectangular box) that would fit around the entire set of wells in thearray of the cassette.

As used herein, the term “assay components” refers to any component thatwould be added to a cell screening assay, including but not limited toreagents, cells, test compounds, media, antibodies, luminescent andreporters.

As used herein, the term “luminescent” encompasses any type of lightemission, including but not limited to luminescence, fluorescence, andchemiluminescence.

In one aspect, the present invention teaches a method of making amicro-array of multiple cell types on a common substrate. As definedherein, a micro-array of multiple cell types refers to an array of cellson a substrate that are not distributed in a single uniform coating onthe support surface, but rather in a non-uniform fashion such that each“cell binding location” or groups of cell binding locations on thesubstrate may be unique in its cell binding selectivity.

The method of making a micro-array of multiple cell types comprisespreparing a micro-patterned chemical array (also referred to herein as achemically modified array of cell binding locations), chemicallymodifying the array non-uniformly, and binding cells to the non-uniformchemical array.

In a preferred embodiment, a micro-patterned chemical array comprises asubstrate 4 which is treated to produce a hydrophobic surface acrosswhich are dispersed at regular intervals hydrophilic spots or “cellbinding locations” 8. (FIG. 1A-1B). The substrate can be a glass,plastic, or silicon wafer, such as a conventional light microscopecoverslip, but can also be made of any other suitable material toprovide a substrate. As describe previously, the term “cell bindinglocations” is used to describe a specific spot on the substrate, anddoes not require any particular depth. The surface of the substrate 4 ispreferably about 2 cm by 3 cm, but can be larger or smaller. In apreferred embodiment, the cell binding locations 8 of themicro-patterned chemical array contain reactable functional groups suchas, but not limited to, amino hydroxyl, sulfhydryl or carboxyl groupsthat can bind to cells non-specifically or be further chemicallymodified to bind molecules that bind cells specifically.

Chemically modified cell binding locations are produced by specificchemical modifications of the cell binding locations on the substrate.The cell binding locations may comprise a variety of different cellbinding molecules that permit attachment and growth of different typesof cells in the cell binding locations, or may permit attachment of onlya single cell type. The hydrophobic domains surrounding the cell bindinglocations on the substrate do not support the attachment and growth ofthe cells.

In one embodiment, a micro-array of multiple cell types is made bycoating a glass wafer via chemisorbance with a layer of a substancehaving reactable functional groups such as amino groups. In a preferredembodiment, an aminosilane such as 3-amino propyltrimethoxysilane (APTS)or N-(2-aminoethyl-3-aminopropyl)trimethoxysilane (EDA) is used, butother reactable substances may be used. Following this first step, dueto the presence of the reactable functional groups, the entire surfaceof the coated glass wafer is hydrophilic.

Secondly, a micro-patterning reaction is carried out where dropscontaining a substance having photo-cleavable or chemically removableamino protecting groups are placed in a micro-pattern of discretelocations on the aminosilane coated glass wafer. In one embodiment thepattern comprises a rectangular or square array, but any suitablediscrete pattern, may be used (such as, but not limited to, triangularor circular). In one embodiment, the drops range in volume from 1nanoliter (nl) to 1000 nl. In a preferred embodiment the drops rangefrom 250-500 nl in volume. Suitable photochemically removable aminoprotecting substances include, but are not limited to4-bromomethyl-3-nitrobenzene, 1-(4,5-dimethoxy-2-nitrophenyl)-ethyl(DMNPE) and butyloxycarbonyl. In one embodiment, the patterning reactionis carried out for 1 to 100 minutes at temperatures ranging from ambienttemperature to 37° C., using reagent concentrations of between 1micromolar (uM) and 1000 uM. In a preferred embodiment, the reaction iscarried out at 37° C. for 60 minutes using a reagent concentration of500 uM.

The drops may be placed onto the aminosilane coated glass wafer viaconventional ink-jet technology. (U.S. Pat. No. 5,233,369; U.S. Pat. No.5,486,855). Alternatively, an array of pins, defined herein as taperedrods that can transfer between 1 nl and 1000 nl of fluid, is dipped intoa bath of the amino protecting substance to produce drops of theprotecting substance on their ends. The pins are then contacted with theglass wafer to transfer the drops thereto. In another embodiment, anarray of capillary tubes made of glass or plastic, as described in U.S.Pat. Nos. 5,567,294 and 5,527,673, (both herein incorporated byreference), containing the amino protecting substance is contacted withthe glass wafer to transfer the droplets to the surface. Thus, the glasswafer is micro-patterned with an array of spots or cell bindinglocations that contain protected amino groups on a hydrophobic surface(FIG. 2A-B).

Third, a hydrophobic substance reactive with unprotected amino groups iswashed over the glass wafer. The hydrophobic substance can be a fattyacid or an alkyl iodide, or any other suitable structure. Certainconditions for such a derivatization of glass can be found in Prime andWhitesides, Science 252:1164-1167, 1991, Lopez et al., J. Am. Chem. Soc.115:5877-5878, 1993, and Mrksich and Whitesides, Ann. Rev. Biophys.Biomol. Struct. 25:55-78, 1996. The fatty acid or alkyl iodide reactswith the unprotected amino groups and covalently attaches thereto, andthe amino groups are now hydrophobic due to the fatty acid or alkyliodide group. The resulting modified array of cell binding locations 9comprises a glass wafer 4 with an array of cell binding locations 8containing protected amino groups on a hydrophobic background. (FIG.2C).

Fourth, the non-uniform array of cell binding locations is produced byuniformly de-protecting the amino groups in a micro-patterned chemicalarray produced according to the above-described methods. In oneembodiment, chemical specificity can be added by chemically crosslinkingspecific molecules to the cell binding locations. There are a number ofwell known homo- or hetero-bi-functional crosslinking reagents such asethylene glycol bis(succinimidylsuccinate) that will react with the freeamino groups in the cell binding locations and crosslink to a specificmolecule. Reagents and conditions for crosslinking free amino groupswith other biomolecules are well known in the art, as exemplified by thefollowing references: Grabarek and Gergely, Analyt. Biochem 185:131-135,1990; McKenzie et al., J. Prot. Chem. 7:581-592, 1988; Brinkley,Bioconjugate Chem. 3:12-13; 1992, Fritsch et al., Bioconjugate Chem.7:180-186, 1996; and Aplin and Hughes, 1981.

In a preferred embodiment, a non-uniform array of cell binding locationsis produced in combinatorial fashion. The resulting cellbinding-locations are non-uniform (i.e., each cell binding location orgroup of cell binding locations may be unique in its cell bindingselectivity). By the term combinatorial, it is meant that the cellbinding locations are variably treated.

In one embodiment, the protected amino groups of the modified array ofcell binding locations of step 3 are de-protected and then specificmolecules with chemical crosslinking reagents are deposited in a desiredpattern. The specific crosslinking agents can bind to the amino groupsand further possess a cell-binding group. In this step, the type of cellbinding group can be varied from one cell binding location to another,or from one group of cell binding locations to another, to create anon-uniform design in the array.

In another embodiment, the amino groups of the chemically modified cellbinding locations of step 3 are uniformly de-protected. Aphoto-activatable crosslinker is reacted with the de-protected aminogroups. An optical mask of a desired pattern is placed over the surfaceof the cell binding locations and the exposed cell binding locations areilluminated with a light source. The position and number of cell bindinglocations that receive light is controlled by the micro-pattern of theoptical mask. Suitable photo-activatable crosslinkers include arylnitrenes, fluorinated aryl azides, benzophenones, and diazopyruvates.Reagents and conditions for optical masking and crosslinking arediscussed in Prime and Whitesides, 1991; Sighvi et al., 1994, Sigal etal., 1996 and Mrksich and Whitesides, 1996. The photo-activatablecrosslinker is bi-functional in that it chemically bonds to the aminogroup on the cell binding locations and, when exposed to light,covalently bonds to cell binding molecules, such as antibodies. Reagentsand conditions for photoactivated crosslinking are discussed in Theveninet al., Eur. J. Biochem. 206:471-477, 1992 and Goldmacher et al.,Bioconjugate Chem. 3:104-107, 1992.

When a photo-activatable crosslinker is used, the glass plate is floodedwith cell binding molecules to be bound to the cell binding locations.In one embodiment, cell binding molecules such as cell surfaceantigen-reactive antibodies, extracellular matrix proteins, (forexample, fibronectin or collagen) or charged polymers (for examplepoly-L-lysine or poly-L-arginine) are used in concentrations rangingfrom about 0.1 to about 1 mM. While the cell binding molecules cover thecell binding locations, the glass plate is irradiated from the undersideof the glass plate, at an angle below the critical angle of the materialof the glass plate, resulting in total internal reflection of the light.(For discussion of total internal reflection fluorescence microscopy,see Thompson et al., 1993). In one embodiment, the irradiation iscarried out at between ambient temperature and 37° C. for 0.1 to 10seconds with light of wavelength between 300 nanometers (nm) to 1000 nm.In a preferred embodiment, the irradiation is conducted at ambienttemperature for 1 second using light with a wavelength of between about300 and 400 nm. Optical crosslinking limits the photo-activatablecrosslinking to a short distance into the solution above the cellbinding locations, and is described in Bailey et al., Nature 366:44-48,1993; Farkas et al., Ann. Rev. Physiol. 55:785-817, 1993; Taylor et al.,Soc. Opt. Instr. Eng. 2678:15-27, 1996; Thompson et al., in Mason, W. T.(ed.), “Fluorescent and Luminescent Probes for Biological Activity.” SanDiego: Academic Press pp. 405-419, 1993.

The photo-activatable crosslinker binds with the cell binding moleculessuch as antibodies and matrix proteins, only in the cell bindinglocations where the crosslinker was irradiated. For example, a singlerow of an array of cell binding locations can be irradiated to produce asingle row of cell binding locations with cell binding molecules boundto the crosslinker. Following a washing of the array to eliminate anyunbound cell binding molecule, a second row of cell binding locationscan be bound to a second cell binding molecule by subsequent flooding ofthe glass wafer with the second cell binding molecule while irradiatingthe second row and optically masking the other rows. Unbound cellbinding molecules are removed by washing the array with PBS, or anyother suitable buffer. In this fashion, multiple rows of cell bindinglocations or groups of cell binding locations can be sequentiallyilluminated by sequential masking in the presence of a particular cellbinding molecule. Alternatively, each cell binding location can beirradiated one by one using pinpoint exposure and optical masking. Inthis manner, different cell binding molecules are bound to rows of thearray or to individual cell binding locations, creating a non-uniformarray of cells bound to the cell binding locations of any desiredpattern.

In a further embodiment for producing chemically modified arrays of cellbinding locations, a chemically modified array is first produced whereinthe amino groups of the cell binding locations are uniformly protectedwith photo-cleavable protecting groups. Rows, columns, and/or individualcell binding locations are sequentially photo-deprotected to expose thefree amino groups by using an optical mask of various patterns to coverall but the cell binding locations to be de-protected. The exposed cellbinding locations (i.e., those not covered by the mask), areilluminated, resulting in removal of the protecting groups. The array isflooded with a bi-functional crosslinker which chemically bonds to thede-protected amino group and activates the cell binding locations.Conditions for the photode-protection of amino groups are discussed inPadwa, A. (ed.) “Organic Photochemistry.”, New York 9:225-323, 1987, Tenet al., Makromol. Chem. 190:69-82, 1989, Pillai, Synthesis 1980:1-26,1980, Self and Thompson, Nature Medicine 2:817-820, 1996 and Senter etal., Photochem. Photobiol. 42:231-237, 1985. Next, cell bindingmolecules are flooded onto the modified chemical array wherein theyreact with the other half of the crosslinker. The array is then washedto eliminate any unbound bi-functional crosslinker and cell bindingmolecules. Another cell binding location or set of cell bindinglocations may be de-protected using another optical mask, and the arraymay then be flooded with a second treatment of a bi-functionalcrosslinker followed by a distinct cell binding molecule which bonds tothis second cell binding location or set of cell binding location ofde-protected amino groups. The array is washed to eliminate the secondtreatment of a bi-functional crosslinker and cell binding molecules. Anon-uniform array of cell binding molecules may thus be produced by arepeated sequence of photo-de-protection, chemical crosslinking ofspecific molecules and washing under a variety of masks. Alternatively,the crosslinking reagents can be delivered to the de-protected cellbinding locations together with the cell binding molecules in one step.Concentration gradients of attached cell binding molecules can becreated by controlling the number of de-protected amino groups exposedusing an optical mask, or by controlling the dose of irradiation for thephoto-activatable crosslinkers.

The chemically modified array of cell binding locations is then used toproduce a non-uniform array of cells on the cell binding locations. Inone embodiment, the modified chemical array is “seeded” with cells byintroducing suspended cells onto the array, allowing binding of thecells to the cell binding locations and then rinsing the wafer to removeunbound and weakly bound cells. The cells are bound only in the cellbinding locations, because the specific chemical environment in the cellbinding locations, in conjunction with the hydrophobic environmentsurrounding each of the cell binding locations, permits the selectivebinding of cells to the cell binding locations only. Furthermore, themodification of cell binding locations with specific cell-bindingmolecules permits selective binding of cells to specific cell bindinglocations, producing a non-uniform array of cells on the cell bindinglocations. In addition, the cell surface molecules that specificallybind to the cell binding locations may be either naturally present orgenetically engineered by expressing “cell binding location-specific”molecules that have been fused to cellular trans-membrane molecules suchthat cells interact with and bind specifically to modified cell bindinglocations. The creation of an array of cell binding locations withdifferent cell recognition molecules allows one cell binding location, agroup of cell binding locations, or the entire array to specifically“recognize”, grow and screen cells from a mixed population of cells.

In one embodiment, cells suspended in culture medium at concentrationsranging from about 10³ to about 10⁷ cells per ml are incubated incontact with the cell binding locations for 1 to 120 minutes attemperatures ranging from ambient temperature to 37° C. Unbound cellsare then rinsed off of the cell binding locations using culture mediumor a high density solution to lift the unbound cells away from the boundcells. (Channavajjala, et al., J. Cell Sci. 110:249-256, 1997). In apreferred embodiment, cells suspended in culture medium atconcentrations ranging from about 10⁵ to about 10⁶ cells per ml areincubated in contact with the cell binding locations at 37° C. for timesranging from about 10 minutes to about 2 hours.

The density of cells attached to the cell binding locations iscontrolled by the cell density in the cell suspension, the timepermitted for cell attachment to the chemically modified cell bindinglocations and/or the density of cell binding molecules in the cellbinding locations. In one embodiment of the cell attachment procedure,10³- to 10⁷ cells per ml are incubated at between ambient temperatureand 37° C. for between 1 minute and 120 minutes, with cell bindinglocations containing between 0.1 and 100 nmoles per cm² of cell bindingmolecules. In a preferred embodiment, 10⁵ and 10⁶ cells per ml areincubated for 10 minutes to 2 hours at about 37° C., with cell bindinglocations containing about 10 to 100 mmoles per cm² of cell bindingmolecules.

In one embodiment, the cells may be chemically fixed to the cell bindinglocations as described by Bell et al., J. Histochem. Cytochem35:1375-1380, 1987; Poot et al., J. Histochem. Cytochem 44:1363-1372,1996; Johnson, J. Elect. Micros. Tech. 2:129-138, 1985, and then usedfor screening at a later time with luminescently labeled molecules suchas antibodies, nucleic acid hybridization probes or other ligands.

In another embodiment, the cells can be modified with luminescentindicators of cell chemical or molecular properties, seeded onto thenon-uniform chemically modified array of cell binding locations andanalyzed in the living state. Examples of such indicators are providedin Giuilano et al., Ann. Rev. Biophys. Biomol. Struct. 24:405-434, 1995;Harootunian et al., Mol. Biol. Cell 4:993-1002, 1993; Post et al., Mol.Biol. Cell 6:1755-1768, 1995; Gonzalez and Tsien, Biophys. J.69:1272-1280, 1995; Swaminathan et al., Biophys. J. 72:1900-1907, 1997and Chalfie et al., Science 263:802-805, 1994. The indicators can beintroduced into the cells before or after they are seeded onto the arrayby any one or a combination of variety of physical methods, such as, butnot limited to diffusion across the cell membrane (reviewed in Haugland,Handbook of fluorescent probes and research chemicals, 6th ed. MolecularProbes, Inc., Eugene, 1996), mechanical perturbation of the cellmembrane (McNeil et al., J. Cell Biology 98:1556-1564, 1984; Clarke andMcNeil, J. Cell Science 102:533-541, 1992; Clarke et al., BioTechniques17:1118-1125, 1994), or genetic engineering so that they are expressedin cells under prescribed conditions. (Chalfie et al., 1994). In apreferred embodiment, the cells contain luminescent reporter genes,although other types of reporter genes, including those encodingchemiluminescent proteins, are also suitable. Live cell studies permitanalysis of the physiological state of the cell as reported byluminescence during its life cycle or when contacted with a drug orother reactive substance.

In another aspect of the present invention, a non-uniform cell array onthe cell binding locations is provided, wherein cells are non-uniformlybound to a chemically modified array of cell binding locations on asubstrate. The cell array is non-uniform because the underlyingnon-uniform chemically modified array of cell binding locations providesa variety of cell binding sites of different specificity. Any cell typecan be arrayed, providing that a molecule capable of specificallybinding that cell type is present in the chemically modified array ofcell binding locations. Preferred cell types for the non-uniform cellarray on the cell binding locations include lymphocytes, cancer cells,neurons, fungi, bacteria and other prokaryotic and eukaryotic organisms.For example, FIG. 3A shows a non-uniform cell array on the cell bindinglocations containing fibroblastic cells grown on a surface patternedsubstrate and labeled with two fluorescent probes (rhodamine to stainactin and Hoechst to stain nuclei), while FIG. 3B shows a non-uniformcell array on the cell binding locations containing fibroblastic cellgrowth (L929 and 3T3 cells) in spotted patterns, labeled with twofluorescent probes and visualized at different magnifications.

Examples of cell-binding molecules that can be used in the non-uniformcell array on the cell binding locations include, but are not limited toantibodies, lectins and extracellular matrix proteins. Alternatively,genetically engineered cells that express specific cell surface markerscan selectively bind directly to the modified cell binding locations.The non-uniform cell array on the cell binding locations may compriseeither fixed or living cells. In a preferred embodiment, the non-uniformcell array on the cell binding locations comprises living cells such as,but not limited to, cells “labeled” with luminescent indicators of cellchemical or molecular properties.

In another aspect of the present invention, a method for analyzing cellsis provided, comprising preparing a non-uniform cell array on the cellbinding locations wherein the cells contain at least one luminescentreporter molecule, contacting the non-uniform cell array on the cellbinding locations to a fluid delivery system to enable reagent deliveryto the cells, conducting high-throughput screening by acquiringluminescence image of the entire non-uniform cell array at lowmagnification to detect luminescence signals from all cell bindinglocations at once to identify those that exhibit a response. This isfollowed by high-content detection within the responding cell bindinglocations using a set of luminescent reagents with differentphysiological and spectral properties, scanning the selected cellbinding locations to obtain luminescence signals from the luminescentreporter molecules in the cells, converting the luminescence signalsinto digital data and utilizing the digital data to determine thedistribution, environment or activity of the luminescent reportermolecules within the cells.

Preferred embodiments of the non-uniform cell array on the cell bindinglocations are disclosed above. In a preferred embodiment of the fluiddelivery system, a chamber, mates with the substrate containing thenon-uniform cell array. The chamber is preferably made of glass, plasticor silicon, but any other material that can provide a chamber issuitable. One embodiment of the chamber 12 shown in FIG. 4 has an arrayof etched domains 13 matching the cell binding locations 8 on thesubstrate 4. In addition, input channels 14 are etched to supply fluidto the etched domains 13. A series of “output” channels 16, to removeexcess fluid from the etched domains 13, can also be connected to thecell binding locations. The chamber 12 and substrate 10 togetherconstitute a cassette 18. While this embodiment utilizes etched domains,any other type of depression 13 formed at the fluidic location 1 canalso be utilized in this embodiment. Alternatively, the fluidic locationmay be flat and the cell binding locations 8 may comprise depressionsthat match the fluidic locations 1. In another alternative, both thecell binding site 8 and the fluidic location 1 are flat, and a volumespace for the well is created by the use of a spacer support 20 betweenthe substrate 4 and the chamber 12.

The chamber 12 is thus used for delivery of fluid to the cells arrayedon the cell binding locations 10. The fluid can include, but is notlimited to a solution of a particular drug, protein, ligand, or othersubstance which binds with surface expressed moieties of cells or thatare taken up by the cells. The fluid to interact with the cells arrayedon the cell binding locations 10 can also include liposomesencapsulating a drug. In one embodiment, such a liposome is formed froma photochromic material, which releases the drug upon exposure to light,such as photoresponsive synthetic polymers. (Reviewed in Willner andRubin, Chem. Int. Ed. Engl. 35:367-385, 1996). The drug can be releasedfrom the liposomes in all channels 14 simultaneously, or individualchannels or separate rows of channels may be illuminated to release thedrug sequentially. Such controlled release of the drug may be used inkinetic studies and live cell studies. Control of fluid delivery can beaccomplished by a combination of micro-valves and micro-pumps that arewell known in the capillary action art. (U.S. Pat. No. 5,567,294; U.S.Pat. No. 5,527,673; U.S. Pat. No. 5,585,069, all herein incorporated byreference.)

Another embodiment of the chamber 12 shown in FIG. 5 has an array ofinput channels 14 matching the chamber's etched domains 13 which areslightly larger in diameter than the cell binding locations 8 on thesubstrate 4, so that the cell binding sites are immersed into the etcheddomains 13 of the chamber 12. Spacer supports 20 are placed between thechamber 12 and the cells arrayed on the cell binding locations 10 alongthe sides of contact. The substrate 4 and the chamber 12 can be sealedtogether using an elastomer or other sticky coating on the raised regionof the chamber. Each etched domain 13 of the chamber 12 can beindividually or uniformly filled with a medium that supports the growthand/or health of the cells arrayed on the cell binding locations 10. Ina further embodiment (FIG. 6), the chamber contains no input channels,for treating all the cells arrayed on the cell binding locations 10 withthe same solution.

Delivery of drugs or other substances is accomplished by use of variousmodifications of the chamber as follows. A solution of the drug to betested for interaction with cells of the array can be loaded from a 96well microtiter plate into an array of micro-capillary tubes 24. (FIG.7). The array of micro-capillary tubes 24 corresponds one-to-one withthe input channels 14 of the chamber 12, allowing solution to flow or bepumped out of the micro-capillary tubes 24 into the channels 14. Thecassette 18 is inverted so that the cell binding locations 8 becomesubmerged in the etched domain 13 filled with the fluid (FIG. 7B). Oncethe interaction between the fluid and cells occurs, luminescence signalsemanating from the cells arrayed on the cell binding locations 10 can bemeasured directly or, alternatively, the substrate 4 can be lifted offthe chamber for post processing, fixation, and labeling. The placementand removal of the array of cells may be accomplished via roboticsand/or hydraulic mechanisms. (Schroeder and Neagle, 1996)

In one embodiment of the chamber 12 shown in FIG. 7, the channels andmatching etched domains 13 are etched into the chamber chemically (Primeand Whitesides, 1991; Lopez et al., 1993; Mrksich and Whitesides, 1996).The etched domains 13 are larger in diameter than the cell bindinglocations 8. This permits the chamber 12 to be contact sealed to thesubstrate 4, leaving space for the cells and a small volume of fluid.Input channels 14 are etched into each row of etched domains 13 of thechamber 12. Each input channel 14 extends from two opposing edges of thechamber 12 and is open at each edge. The etched domains 13 of a singlerow are in fluid communication with the input channels 14 by placing amicro-capillary tube 24 containing a solution into contact with the edgeof the chamber 12. Each row of connected input channels 14 can be filledsimultaneously or sequentially. During filling of the input channels 14by valves and pumps or capillary action, each of the channels of thechamber 12 fills and the drug passes to fill each etched domain 13 inthe row of etched domains 13 connected by the input channel 14.

In a further embodiment of the chamber 12, raised reservoirs 28 andinput channels 14 can be placed onto the surface of the chamber 12 asshown in FIG. 8 b. In a preferred embodiment, the raised reservoirs 28and input channels 14 can be made from polytetrafluoroethylene orelastomeric material, but they can be made from any other stickymaterial that permits attachment to the substrate 4, such aspoly(dimethylsiloxane), manufacture by Dow Corning under the trade nameSYLGARD 184™. The effect is the same as with a chamber having etchedchannels and channels and its uses are similar.

In another embodiment of the chamber shown in FIG. 8A, a first channel30 extends from one edge of the chamber 12 to a first etched domain 13or raised reservoirs 28 and channels. A second channel 32 extends fromthe opposing edge to a second etched domain adjacent the first etcheddomain. The first 30 and second 32 channels are not in fluidcommunication with each other yet are in the same row of input channels14 or raised reservoirs 28.

In another embodiment, as shown in FIGS. 9 and 10, the chamber 12 mayhave an input channel 14 extending from each etched domain 13 or raisedreservoir 28 to the edge of the chamber. The channels 14 can alloriginate from one edge of the chamber 12 (FIG. 9), or from both edges(FIG. 10). The input channels 14 can also be split to both sides of theetched domains 13 to minimize the space occupied by the input channels14. Separate fluidic channels allow for performance of kinetic studieswhere one row at a time or one depression at a time is charged with thedrug.

In a further embodiment depicted in FIG. 11, each etched domain 13 is influid communication with a corresponding input channel 14 having a plug36 between the end of the channel 14 and the etched domain 13, whichprevents the injected solution from flowing into the etched domain 13until the desired time. Solutions may be preloaded into the inputchannels 14 for use at a later time. A plug 36 likewise can be disposedbetween a terminal etched domain 13 in a set of connected etched domains13 in fluid communication with an input channel 14. Upon release of theplug 36, the substance flows through and fills all the etched domains 13which are in fluid communication with the input channel 14.

In one embodiment, the plugs 36 are formed of a hydrophobic polymer,such as, but not limited to proteins, carbohydrates or lipids that havebeen crosslinked with photo-cleavable crosslinkers that, uponirradiation, becomes hydrophilic and passes along with the drug into thedepression. Alternatively, the plug 36 may be formed of a crosslinkedpolymer, such as proteins, carbohydrates or lipids that have beencrosslinked with photo-cleavable crosslinkers that, when irradiated,decomposes and passes into the etched domain 13 along with the solution.

The cassette 18, which comprises the substrate 4 and the chamber 12, isinserted into a luminescence reader instrument. The luminescence readerinstrument is an optical-mechanical device that handles the cassette,controls the environment, controls delivery of solutions to wells, andanalyzes the luminescence emitted from the array of cells, either onewell at a time or the whole array simultaneously. In a preferredembodiment (FIG. 12), the luminescence reader instrument 44 comprises anintegrated circuit inspection station using a fluorescence microscope asthe reader and microrobotics to manipulate the cassettes. The reader ofthe present invention can comprise any optical system designed to imagea luminescent specimen onto a detector. A storage compartment 48 holdsthe cassettes 18, from where they are retrieved by a robotic arm 50 thatis controlled by computer 56. The robotic arm 50 inserts the cassette 18into the luminescence reader instrument 44. The cassette 18 is removedfrom the luminescence reader instrument 44 by another robotic arm 52,which places the cassette 18 into a second storage compartment 54.

The luminescence reader instrument 44 is an optical-mechanical devicedesigned as a modification of light optical-based, integrated circuitinspection stations used to “screen” integrated circuit “chips” fordefects. Systems integrating environmental control, micro-robotics andoptical readers are produced by companies such as Carl Zeiss [Jena,GmbH]. In addition to facilitating robotic handling, fluid delivery, andfast and precise scanning, two reading modes, high content and highthroughput are supported. High-content readout is essentially the sameas that performed by the ARRAYSCAN™ reader (U.S. Pat. No. 5,989,835). Inthe high content mode, each location on the micro-array of multiple celltypes is imaged at magnifications of 5-40× or more, recording asufficient number of fields to achieve the desired statisticalresolution of the measurement(s).

In the high throughput mode, the luminescence reader instrument 44images the micro-array of multiple cell types at a much lowermagnification of 0.2× to 1.0× magnification, providing decreasedresolution, but allowing all the cell binding locations on the substrateto be recorded with a single image. In one embodiment, a 20 mm×30 mmmicro-array of multiple cell types imaged at 0.5× magnification wouldfill a 1000×1500 array of 10 um pixels, yielding 20 um/pixel resolution,insufficient to define intracellular luminescence distributions, butsufficient to record an average response in a single well, and to countthe numbers of a particular cell subtype in a well. Since typicalintegration times are on the order of seconds, the high-throughput modeof reading technology, coupled with automated loading and handling,allows for the screening hundreds of compounds a minute.

In one embodiment shown in FIG. 13, the luminescence reader instrumentcomprises an optical-mechanical design that is either an upright orinverted fluorescence microscope 44, which comprises acomputer-controlled x,y,z-stage 64, a computer-controlled rotatingnosepiece 68 holding a low magnification objective 70 (e.g., 0.5×) andone or more higher magnification objectives 72, a white light sourcelamp 74 with excitation filter wheel 76, a dichroic filter system 78with emission filters 80, and a detector 82 (e.g., cooled charge-coupleddevice). For the high throughput mode, the low magnification objective70 is moved into place and one or more luminescence images of the entirecell array is recorded. Wells that exhibit some selected luminescenceresponse are identified and further analyzed via high content screening,wherein the nosepiece 68 is rotated to select a higher magnificationobjective 72 and the x,y,z-stage 64 is adjusted to center the “selected”well for both cellular and subcellular high content screening, asdescribed in U.S. Pat. No. 5,989,835.

In an alternate embodiment, the luminescence reader instrument 44 canutilize a scanned laser beam in either confocal or standard illuminationmode. Spectral selection is based on multiple laser lines or a group ofseparate laser diodes, as manufactured by Carl Zeiss (Jena, GmbH,Germany) or as discussed in Denk, et al. (Science 248:73, 1990).

Another embodiment of the high throughput screening mode involves theuse of a low-resolution system consisting of an array (1×8, 1×12, etc.)of luminescence exciters and luminescence emission detectors that scanssubsets of the wells on a non-uniform micro-patterned array of cells. Ina preferred embodiment, this system consists of bundled optical fibers,but any system that directs luminescence excitation light and collectsluminescence emission light from the same well will suffice. Scanningthe entire micro-array of multiple cell types with this system yieldsthe total luminescence from each well, both from cells and the solutionthey are bathed in. This embodiment allows for the collection ofluminescence signals from cell-free systems, so-called “homogeneous”assays.

FIG. 14A shows a method, in the form of a flow chart, for analyzing amicro-array of multiple cell types in both the high throughput and highcontent modes using the luminescence reader instrument, which first useshigh throughput detection to measure a response from the entire array“A”. (FIG. 14B). Any well that responds above a preset threshold isconsidered a hit and the cells in that well are measured via highcontent screening. (FIG. 14C). The high content mode (“B”) may or maynot measure the same cell parameter measured during the high throughputmode (“A”).

In another aspect of the invention, a cell screening system isdisclosed, wherein the term “screening system” comprises the integrationof a luminescence reader instrument, a cassette that can be insertedinto the luminescent reader instrument comprising a micro-array ofmultiple cell types wherein the cells contain at least one luminescentreporter molecule and a chamber associated with the non-uniformmicro-patterned array of cells, a digital detector for receiving datafrom the luminescence reader instrument, and a computer means forreceiving and processing digital data from the digital detector.

Preferred embodiments of the luminescence reader instrument, and thecassette comprising the micro-array of multiple cell types and thechamber are disclosed above. A preferred embodiment of the digitaldetector is disclosed in U.S. Pat. No. 5,989,835, and comprises a highresolution digital camera that acquires luminescence data from theluminescence reader instrument and converts it to digital data. In apreferred embodiment, the computer means comprises a digital cable thattransports the digital signals from the digital detector to thecomputer, a display for user interaction and display of assay results, ameans for processing assay results, and a digital storage media for datastorage and archiving, as described in U.S. Pat. No. 5,989,835.

In a preferred embodiment, the cell screening system of the presentinvention comprises integration of the preferred embodiments of theelements disclosed above (FIG. 15). The array of multiple cell types 10comprises cells bound to chemically modified cell binding locations 8 ona substrate 4. The chamber 12 serves as a microfluidic delivery systemfor the addition of compounds to the cells 10 on the substrate 4, andthe combination of the two comprises the cassette 18. The cassette 18 isplaced in a luminescence reader instrument 44. Digital data areprocessed as described above and in U.S. Pat. No. 5,989,835, herebyincorporated by reference in its entirety. The data can be displayed ona computer screen 86 and made part of a bioinformatics database 90, asdescribed in U.S. Pat. No. 5,989,835. This database 90 permits storageand retrieval of data obtained through the methods of the invention, andalso permits acquisition and storage of data relating to previousexperiments with the cells. An example of the computer display screen isshown in FIG. 16.

EXAMPLE 1 Coupling of Antibodies to Micro-Array of Multiple Cell Typesfor the Attachment of Specific Lymphoid Cells

-   1. The cell line used was a mouse B cell lymphoma line (A20) that    does not express IgM on its surface. A micro-array of multiple cell    types was prepared for derivatization by being immersed overnight in    20% sulfuric acid, washed 2-3 times in excess distilled water,    rinsed in 0.1M sodium hydroxide and blotted dry. The micro-array of    multiple cell types was either used immediately or placed in a clean    glass beaker and covered with parafilm for future use.-   2. The micro-array of multiple cell types was placed in a 60 mm    petri dish, and 3-Aminopropyltrimethoxysilane was layered onto the    micro-array of multiple cell types ensuring complete coverage    without running over the edges (approximately 0.2 ml for a 22×22 mm    non-uniform micro-patterned array of cells, and approximately 0.5 ml    for a 22×40 mm non-uniform micro-patterned array of cells). After 4    minutes at room temperature, the micro-array of multiple cell types    was washed in deionized water and excess water was removed by    blotting.-   3. The micro-array of multiple cell types was placed in clean 60 mm    petri dishes and incubated with glutaraldehyde (2.5% in PBS,    approximately 2.5 ml) for 30 minutes at room temperature, followed    by three PBS washes. Excess PBS was removed by blotting.-   4. Cell nuclei in the micro-array of multiple cell types were    labeled with a luminescent Hoechst dye during the blocking step. The    appropriate number of lymphoid cells (see below) in C-DMEM were    transferred to a 15 ml conical tube, and Hoechst dye was added to a    final concentration of 10 μg/ml. Cells were incubated for 10-20    minutes at 37° C. in 5% CO₂, and then pelleted by centrifugation at    1000×g for 7 minutes at room temperature. The supernatant containing    unbound Hoechst dye was removed and fresh media (C-DMEM) was added    to resuspend the cells as follows: approximately 1.25-1.5×10⁵ cells    in 0.2 ml per 22×22 mm non-uniform micro-patterned array of cells,    and approximately 2.5×10⁵ cells in 0.75 ml for the 22×40 mm    non-uniform micro-patterned array of cells.-   5. The micro-array of multiple cell types was washed briefly in PBS    and transferred to a clean, dry 60 mm petri dish, without touching    the sides of the dish. Cells were carefully pipetted onto the top of    the micro-array of multiple cell types at the density noted above.    Dishes were incubated at 37° C. in 5% CO₂ for 1 hour. Unbound cells    were then removed by repeated PBS washings.-   6. Antibody solutions (Goat Anti-Mouse IgM or Goat Anti-Mouse Whole    Serum) were spotted onto parafilm (50 μl for 22×22 mm non-uniform    micro-patterned array of cells, 100 μl for a 22×40 mm non-uniform    micro-patterned array of cells). The micro-array of multiple cell    types was inverted onto the spots, so that the antiserum covered the    entire surface of the treated micro-array of multiple cell types    without trapping air bubbles. The micro-array of multiple cell types    was incubated with the antibody solution for 1 hour at room    temperature.-   7. The micro-array of multiple cell types was carefully lifted from    the parafilm, placed in a clean 60 mm petri dish, and washed three    times with PBS. Unreacted sites are then blocked by the addition of    2.5 ml of 10% serum (calf or fetal calf serum in DMEM or Hank's    Balanced Salt Solution) for 1 hour at room temperature.-   8. Both cell lines should bind to the anti-mouse whole serum, but    only the X16s should bind to the anti-mouse IgM. The binding of    specific lymphoid cell strains to the chemically modified surface is    shown in FIG. 17. The mouse lymphoid A20 cell line, lacking surface    IgM molecules but displaying IgG molecules, bound much more strongly    to the surface modified with whole goat anti-mouse serum (FIG. 17C)    than to the surface modified with goat anti-mouse IgM (FIG. 17B) or    an uncoated slide (FIG. 17A).

EXAMPLE 2 High-Content and High Throughput Screen

The insulin-dependent stimulation of glucose uptake into cells such asadipocytes and myocytes requires a complex orchestration of cytoplasmicprocesses that result in the translocation of GLUT4 glucose transportersfrom an intracellular compartment to the plasma membrane. A number ofmolecular events are triggered by insulin binding to its receptor,including direct signal transduction events and indirect processes suchas the cytoskeletal reorganizations required for the translocationprocess. Because the actin-cytoskeleton plays an important role incytoplasmic organization, intracellular signaling ions and moleculesthat regulate this living gel can also be considered as intermediates ofGLUT4 translocation.

A two level screen for insulin mimetics is implemented as follows. Cellscarrying a stable chimera of GLUT4 with a Blue Fluorescent Protein (BFP)are arranged on the micro-array of multiple cell types arrays, and thenloaded with the acetoxymethylester form of Fluo-3, a calcium indicator(green fluorescence). The array of locations are then simultaneouslytreated with an array of compounds using the microfluidic deliverysystem, and a short sequence of Fluo-3 images of the whole micro-arrayof multiple cell types are analyzed for wells exhibiting a calciumresponse in the high throughput mode. The wells containing compoundsthat induced a response, are then analyzed on a cell by cell basis forevidence of GLUT4 translocation to the plasma membrane (i.e., thehigh-content mode) using blue fluorescence detected in time and space.

FIG. 18 depicts the sequential images of the whole micro-array ofmultiple cell types in the high throughput mode (FIG. 18A) and the highcontent mode (FIG. 18B). FIG. 19 shows the cell data from the highcontent mode.

EXAMPLE 3 Improved Cassettes and Enhanced Well Density

In another aspect, the present invention provides devices and methodsfor maximizing the cell-plated area and the number of wells that can beimaged in a sub-array, while still obtaining adequate pixel resolutionin the image. This result has been achieved through the use of fluidicarchitectures that minimize the distance and area between wells, thusmaximizing well density.

In one embodiment of this aspect is provided a cassette for cellscreening comprising a substrate having a surface, wherein the surfacecontains a plurality of cell binding locations; a fluid delivery systemfor delivering reagents to the plurality of cell binding locations,wherein the fluid delivery system comprises a multi-level chamber thatmates with the substrate, wherein the multi-level chamber comprises

-   -   i. a crossed array of microfluidic input channels and output        channels, wherein each well is in fluid connection with one or        more input channels and one or more output channels;    -   ii. a plurality of fluidic locations in fluid connection with        the microfluidic input channels and output channels;    -   iii. one or more input manifolds in fluid connection with the        microfluidic input channels;    -   iv. one or more output manifolds in fluid connection with the        microfluidic output channels;    -   v. at least one source receptacle in fluid connection with the        one or more input manifolds; and    -   vi. at least one waste receptacle in fluid connection with one        or more output manifolds; and

a plurality of wells, wherein an individual well comprises the spacedefined by the mating of one cell binding location and one fluidiclocation.

In preferred embodiments, the cassettes further comprise a pump tocontrol fluid flow within the microfluidic device; a substratetemperature controller and/or a controller to regulate oxygen and carbondioxide partial pressures within the device.

The substrate of the present invention is chemically modified so thatcells selectively adhere to a cell growth substrate and are containedwithin small regions, referred to as “cell binding locations”, which aresub-millimeter to a few millimeters in size. The combination of a cellbinding location on the substrate and a fluidic location on the chamberdefines a space referred to as a “well”. The substrate may bepredominantly flat and the cell binding locations may be matched withfluidic location depressions on a substrate cover, so that when the twoparts are assembled there is some depth in the area of the wells soformed. Conversely, the substrate cover fluidic location may be flat andthe substrate may contain depressions at the cell binding location, sothat when the two parts are assembled there is some depth in the area ofthe wells so formed. Fluids and test compounds are supplied to the wellsvia a chamber, which combines with the substrate to form a cassette. Ina preferred embodiment, the chamber acts as the substrate cover andcontains fluidic locations in fluid connection with the microfluidicchannels of the device, wherein the fluidic locations comprisedepressions that match the cell binding locations on the substrate, suchthat the volume of each well is etched into the chamber.

On the surface of the substrate, there are sealing regions that liebetween and around the cell binding locations, and there arecorresponding sealing regions on the bottom face of the chamber that liebetween and around the fluidic locations. When the substrate and thechamber are joined, the sealing regions of both components meet to forma seal that prevents the flow of fluid across the sealing regionsbetween the wells thereby formed. These sealing regions of bothcomponents may be physically and/or chemically modified to enhancesealing. In a first example, a hydrophobic silane coating ofoctadecyltrichlorosilane is coated on the sealing regions. In a secondexample, light-curable adhesives and cyanoacrylates that are USP ClassIV-compliant such as are available from Dymax Corporation are amenableto use with biomedical devices made from ceramic, glass, plastic, ormetal. In a third example, a biocompatible tape material with precisioncut-outs that match the wells of the array is available from AveryDennison Specialty Tape Division. Similarly, biomedical acrylicadhesives can be transferred from tapes to surfaces, such as areavailable from Tyco Corporation, for applying an adhesive coatingprecisely to these sealing regions. In a fourth example, a light-curablesilicone elastomer can be used for bonding and sealing between thesealing regions of the substrate and the chamber, such as are availablefrom Master Bond, Inc.

The microfluidic cassette of the present invention has advantages overprior art cell array microfluidic devices used in high content screeningsystems including, but not limited to:

-   1) faster imaging of a statistically-relevant set of cells (based on    imaging a set of cell binding locations (i.e.: “sub-arrays”)    simultaneously; See FIG. 20)-   2) more wells per plate and less physical space occupied per plate    (i.e.: higher well density),-   3) more efficient use of valuable test compounds, and-   4) more efficient use of cells compared to microplates in which the    entire well area is not imaged.

As discussed supra, prior microwell plate fluid exchange for each wellcan only be achieved by means of a pipette being inserted into the welland either ejecting or aspirating fluid to or from the well,respectively. Automated fluid handling in prior microwell plates isachieved by using robotic pipettors that insert a pipette into each wellfor each fluid transfer step in a procedure. This pipette is typicallyrequired also to move to or from another microplate well for the sourceor destination of the fluid that is exchanged with the microwell plateof interest.

Integrated fluidics is advantageous for arrays with sub-millimeterinter-well distances because it is unwieldy, if not impossible, topipette fluids with an acceptable degree of spatial resolution andaccuracy. An acceptable level of accuracy requires that the measurementerror should not exceed 2%. Thus, for a 100 microliter (μl) volume inthe well of a 96 well plate, a measurement error of 2 μl is acceptable.For a 100 nano-liter (nl) volume, a 2 nl measurement error would berequired. This level of accuracy is not reliably available with priorautomatic pipettors across a spectrum of compositions and viscosities.

The minimization of the inter-well distances enables the fast parallelreading of sub-arrays of wells. If the integrated fluidics is too bulky(i.e.: does not allow close-spacing of wells), then such fast parallelreading capability is lost. In a preferred embodiment of the presentinvention, a crossed channel architecture is utilized, allowing forfewer channels, valves, and pumps, and thereby further reducing thespace taken by channels on the cassette of the invention. In a mostpreferred embodiment, the channels are placed in levels above that ofthe wells, permitting the closest possible inter-well spacing. In afurther preferred embodiment, the use of porous medium as a drain ‘pump’allows for simpler design and fabrication of the system. According tothe invention, the channels may be of any size that permits the fluidicarchitecture herein defined. In a preferred embodiment, they range insize from between about 0.025 mm to about 0.5 mm in width for squarecross-section channels, or in diameter for circular cross-sectionchannels.

The fluidic architecture of the instant microfluidic cassette enablesthe controlling elements (including, but not limited to pumps,manifolds, controlled-pressure vessels, and/or valves) to be locatedoutside the matrix of the cell array. FIG. 40 shows one embodiment ofthe pumping and valving schemes of this invention as it would appearwith all the pumps and valves on-board the cassette. All variations ofthe pumping and valving schemes shown in various Figures (and othersthat would be apparent to those skilled in the art) may be implementedin this way, either with on-board active devices outside the matrix ofthe wells, with off-board active devices, or with some devices on-boardand some devices off-board. As a result, the controlling elements can beeither within the cassette (on-board) or external to the cassette(off-board). In either case, there are no active components within thematrix of the array of wells that might limit the close-spacing ofchannels and wells. Because this cassette is particularly designed forthe culture and analysis of living cells, the fluidic architecture andall of its sub-components and functional parts are compatible with,support, and enable the culture of living cells. The particular aspectsof these sub-components and functional parts that are designed for livecell culture are identified below.

In a preferred embodiment, the cassette of the present inventioncomprises an off-board pump to provide pressure-driven flow that alsocontrols which wells are addressed by the flow. This design featureensures that the pumping method, unlike many prior art methods, is notineffective or harmful when used with cell culture medium that isaqueous, polar and contains proteins and salts. For example,electrohydrodynamic pumping is ineffective with polar solvents (MarcMadou, Fundamentals of Microfabrication, CRC Press, Boca Raton, 1997, p.433). Electro-osmosis is typically accompanied by some degree ofelectrophoretic separation of charged medium components, such asproteins. Also, electro-osmosis typically requires the use of electricfield strengths within the range of 100 V/cm to 1000 V/cm, which mayaffect the physiology of the living cells in the device. The fluidcontrol system of the present device does not suffer from thedisadvantages of electrically-induced methods when using biologicalfluids.

In another embodiment, the pump is on-board and may utilizeelectrically-driven flow, but the electric fields are limited to regionsexternal to the matrix of the array of wells, and no electric fieldgradients are applied across any of the wells in which live cells arepresent. This embodiment is shown in FIG. 41, where two electrodes areused to apply electric potential differences along segments of fluidicchannels corresponding to different on-board source reservoirs (730).Each channel segment with its electrode pair form an electrokinetic pump(740). These source reservoirs (730) may be supplied with media and/orcompounds via fill ports (700) in the cassette surface. Valves (720)between the reservoirs and the pumps are closed whenever thecorresponding electrokinetic pump is off, and are open whenever thecorresponding electrokinetic pump is on, to maintain back pressure andto allow intake of air or fluid, respectively. Control of the voltageapplied to electrode 1 (760) is used to induce electrokinetic flow ofthe fluid in the corresponding channel segment. Electrode 2 (780)(nearest to the matrix of wells) is kept at ground potential, as is thematrix of the wells, to ensure that minimal or no potential differenceand/or electric field is developed in the vicinity of the cells, becauseelectric fields are known to affect cell physiology. Alternatively, theelectrokinetic pump may be located down stream from the array to serveas a negative pressure pump. In this embodiment, electrophoresis of themedia by the pumping action, if any, would only affect the media afterit has been already used by the cells and is on its way to the wastereservoir.

In all embodiments, the use of on-board or off-board pumps that areexternal to the matrix of wells to control fluid flow eliminates theneed for active valves within the matrix of wells, and thus eliminatesthe problem of allocating space within the matrix that might prevent theclose spacing of the wells.

The control of the fluid pathway by means of pressure control alsoenables the optional use of various diffusion control means that arecompatible and effective with cell culture medium that is aqueous, polarand contains proteins and salts. These means also require minimal spaceon the device, and thus do not prevent the close-spacing of wells.

U.S. Pat. No. 5,603,351 describes a microfluidic device that includescrossed channels, but the channel network is not defined to allow two ormore reagents to be combined in a reaction well, but rather to allowreagents to be fed to a well in a serial fashion. The purpose of thepresent cassette is not to expose two or more different fluids to eachother, but to expose the living cells cultured on the well bottoms inserial fashion to two or more different fluids.

The fluidic channels of the cassette of the present invention providetransport of fluid and compounds from source receptacles to each welland from each well to one or more waste receptacles. Each well could beprovided with a separate input and output channel (FIG. 21), yielding2n² channels (100, 120) for an n×n array (040). To control the flow of mliquid or vapor mixtures to an n×n array, m+n² valves or pumps arerequired (FIG. 22). However, for a large array, the number of channels,pumps, and valves for this type of architecture becomes unwieldy. A pumpthat has variable or switchable pressure (e.g., a syringe pump) isfunctionally equivalent to a pressurized reservoir with a valve.Therefore, in the following discussions of pumps, reservoirs, andvalves, it should be understood that these components may be substitutedwithout altering the spirit of the invention.

Crossed-Channel Multilevel Architecture

A preferred embodiment of architectures of the cassette consisting of acrossed, nonintersecting array of input (100) and output (120) channelsand utilizing multiple levels is disclosed. Specific embodiments withinthis class of architectures are given in FIGS. 23-26 and in FIGS. 33 and34.

Because architectures in this class require fewer independent channelsper well compared to the class of architectures with separate input andoutput channels for each well, these crossed-channel architectures alsominimize the lateral space (140) that is required between wells toaccommodate the pathways for the channels.

The crossed-channel, multilevel architectures require only 2n channelsto address n² wells. In a preferred embodiment, the row and columnchannels themselves are formed in two distinct layers so that they donot intersect. The row channels lie above the column channels, or viceversa. These two layers lie either in the same plane of the well layer(FIGS. 23 and 24), or lie in a plane above the well layer (FIGS. 25-26and 33-34). In other embodiments, structures with more than two planesare contemplated.

In a most preferred embodiment, the channels are placed above the planeof the wells, so that the minimum inter-well distance (140) isconstrained only by the optical and physical requirements for a certainwall thickness separating each well. The advantage of placing thechannels in levels above the wells is shown by the following examples.Wells that are 0.4 mm×0.4 mm can be placed on a 0.5 mm pitch grid withallowance for a 0.1 mm wall between wells, yielding a 5 mm×5 mm area fora 10×10 well array. Row and column channels of 0.2 mm width can easilybe formed in upper levels of the device with ample distance between thechannels. By contrast, if 0.2 mm channels with 0.1 mm walls are requiredwithin the plane of the wells (and row and column channel layers areformed in two separate planes within the plane of the wells), the wellpitch increases to 0.8 mm. This yields an 8 mm×8 mm area for a 10×10well array. In this example, the placement of the channels between thewells increases the area required for the array by a factor of 2.6. Fordifferent channel architectures with separate input and output channelsfor each well, the additional space required between wells is muchgreater.

Either for channels above or within the level of the wells, horizontal(160) or vertical (180) connecting channels or vias are formed from eachwell (j,k) to its corresponding row channel j and column channel k.Fabrication of such structures from glass, semiconductor, or plasticmaterials is well known in the art. In one such method, silicon dioxide(SiO₂) is lithographically formed within silicon nitride (Si₃N₄) layersusing a multistep layer-by-layer process. (Turner and Craighead, Proc.SPIE 3528:114-117 (1998)) Exposure of the structure to a wet etchantremoves the SiO₂ to create the channel network within the Si₃N₄, whichis not etched.

Control of Flow in the Crossed-Channel Architecture

In a crossed array of rows (A, B, C, etc.) and columns (1, 2, 3, etc.)the flow of fluid to and from well (j,k) is produced when, for example,positive pressure is applied to row j and the valve of output channel kis opened, where j and k refer to, e.g., row D and column 7,respectively. Flow to all wells of an entire row j is produced when, forexample, positive pressure is applied to row j and the valves of alloutput channels (1, 2, 3, etc.) are opened. A similar procedure wouldyield flow to all wells of a column.

The pressures that are discussed here are actually pressure differencesrelative to ambient atmospheric pressure, otherwise known as “gauge”pressure. Thus, the value of the positive pressure is the amount bywhich an applied pressure is greater than atmospheric pressure; thevalue of the negative pressure is the amount by which an appliedpressure is less than atmospheric pressure. These pressures are referredto as positive and negative gauge pressure, respectively.

The term “fluid pathway” is used herein indicates the particular set ofwells {(j,k), (j,n), (m,k), (m,n) . . . etc.} that is subjected to fluidflow by the enabling of the flow control devices of rows j and m, etc.,and columns k and n, etc. Note that enabling the flow control devices oftwo rows and two columns yields a fluid pathway through four wells. Thisis defined herein as a single fluid pathway involving four wells, ratherthan four pathways involving single wells.

Reduction in the Number of Active Valves or Pumps

Various combinations of actively-controlled flow control devices (pumpsor pressurized reservoirs with valves) are used to produce flow througha desired well (010) or fluid pathway. For example, m compounds can bemultiplexed to a single well by using m positive pressure sourcereservoirs or pumps (200), a valve-less manifold (240) and valve-freewaste reservoir at atmospheric pressure (300)(FIG. 27). This valve-lessmanifold (240) can be replaced by a valved manifold [e.g., (260) of FIG.28] to better control the diffusion of fluids between the m sources.Alternatively, the same functionality can be achieved by using anegative pressure waste reservoir or pump (320) with m valves (260)connected to m source reservoirs at atmospheric pressure (220)(FIG. 28).The use of source reservoirs at atmospheric pressure allows convenientbubbling of gases through the source reservoirs to establish andmaintain desired levels of dissolved CO₂ and O₂ in the media.

In another embodiment, a waste reservoir filled with a porous mediumserves as a capillary action pump (340) and provides negative pressure(FIG. 28). The thermodynamics of wetting creates a pressure across aliquid-vapor interface bounded by a solid-liquid-vapor contact line.This pressure of capillary action can be used to draw a column of liquidfrom any microchannel pathway into the high surface area porous medium.A series of such negative pressure reservoirs can be linked to the arrayvia a multiplexing valved manifold for control of flow, or one singlereservoir can be closely coupled to the output channels of all wellssimultaneously. A number of porous media are known in the art,including, but not limited to silica gel technology, porous ceramicmaterials such as zeolite, a pad of hydrophilic (synthetic or natural)fibers, and partially hydrated or lyophilized hydrogels such asalginates, poly(vinyl) alcohol gels, agarose, sugar polyacrylate gels,and polyacrylamide gels. In a preferred embodiment, the porous medium isbased on silica gel technology. A thin membrane or filtering sheet maybe used to partition the porous media from the microchannel array. Thismethod can provide pressure for pumping until the internal surface areaof the porous medium is largely wetted by the liquid. By suitable choiceof porous medium and design of this method, adequately large amounts ofliquid can be pumped to conduct a chosen measurement with the arraysystem of the present invention.

In another embodiment, the array of m positive pressure sourcereservoirs or pumps (200) shown in FIG. 27 is multiplexed to an n×ncrossed channel array of wells (040) by means of 1×n input (400) and n×1output (410) valve manifolds (FIG. 29). Recall that for architectureswith separate input and output channels for each well (FIG. 22), m+n²valves or pumps are required in this situation. Again, the valve-lessmanifold (240) can be replaced by a valved manifold [e.g., (260) of FIG.28] to better control the diffusion of fluids between the m sources.Flow through well (j,k) is effected when input valve j and output valvek are opened. Alternatively, the same functionality can be achieved withm reservoirs at atmospheric pressure (220) and a negative pressure wastereservoir (either 320 or 340) as in FIG. 28 by means of 1×n input (400)and n×1 output valve (410) manifolds (FIG. 30).

Rinse Channels

In a preferred embodiment, an n×n microwell array is augmented by anadditional column channel k* or set of column channels(s) {k*} thatallow passing of a segment of liquid or vapor from a given row j to awaste reservoir without going through the wells {(j,k)} or the columnchannels {k}. These additional column channels {k*} are referred to asrinse channels (130), because they enable the rinsing of the rowchannels without requiring flow through the wells.

In general, a rinse channel k* (130) can be located adjacent andparallel to column channel k (016) but be linked by a via to row channelj (014) at the point were they cross [and not be linked directly to thecorresponding well (j,k)]. In one embodiment, every column channel k hasan associated parallel rinse channel k* (FIGS. 33 and 34). In otherembodiments, there is only one rinse channel to provide rinsing for theentire set of row channels {j}, or there is any combination of rinsechannels {k*} interspersed within the array of column channels {k}. Whenthere is only one rinse channel, it may be preferred to have it locatedadjacent to the last column channel of the array, where k=n.

FIG. 35 shows, in a case of fluid flow through well (j,k), an example ofa method to rinse a segment of liquid or vapor from row channel j andinto rinse channel k*, where k* is immediately adjacent to columnchannel k. This procedure and architecture allows this fluid segment tobe directed to the waste reservoir without passing over wells {(j, k+1),(j, k+2), etc.} that are downstream along row channel j. This isdesirable to limit the diffusion of a given compound from one well (j,k)to other wells downstream {(j, k+1), (j, k+2), etc.} where the compoundmay not be required. Additionally, as described below, diffusion may becontrolled by introducing vapor segments into the microchannel microwellarray.

FIG. 32 shows how the pumping and valving scheme of FIG. 29, employingpositive pressure source reservoirs, can be extended to work with acrossed array of n row channels and 2n column channels (n ordinarycolumn channels plus n rinse column channels). This requires a 2n×1output valve manifold (420). In general, for every additional rinsechannel k* that is added, one additional valve is added to the valvemanifold connected to the column channels. Similarly, the scheme of FIG.30, employing a negative pressure waste reservoir, can be extended towork with an array incorporating rinse channels (130).

Dual-Pumped Designs

Flow through a fluid pathway {(j,k), (j,n), (m,k), (m,n) . . . etc.} ormore generally through any length of tubing is caused by a continuouspressure gradient that exists along the length of the pathway or tubing.Control of this pressure gradient by means of pressures applied at theinputs and outputs of the fluid pathways requires that there not beleakage of flow across the walls of the pathway. Leakage alsocompromises the proper dosing of compounds to the wells for drugscreening. The sealing of the walls of the pathways must be adequate forthe gauge pressures that are likely to be applied. For fluid flow ratesof roughly a few millimeters per minute, path lengths not greater than afew centimeters, and channel cross sections on the order of 100 mm×100mm, the Hagen-Poiseuille law predicts that the required end-to-endpressure differential ΔP is less than 1 atmosphere (1 atm.≈14 pounds persquare inch). Nevertheless, considering that various materials andstructures known in the art can be implemented for the device of theinvention, the possibility of leakage between neighboring wells isminimized by reducing the gauge pressure exerted across the walls of thesystem. Towards this end, the present device can use positive pressuresource reservoirs with a negative pressure waste reservoir (i.e., a“double-pumped” system) to essentially cut in half the maximum gaugepressures which must be applied to achieve a given total end-to-endpressure differential.

For a “single-pumped” system, i.e., a system that is open to atmosphericpressure at one end and that is pumped at the other end (either to apositive or to a negative gauge pressure, ±ΔP), the pressure differenceexerted across the walls of the system is a maximum at the location ofthe pumped reservoir, i.e., (200) of FIGS. 27 and 29; or (320, 340) ofFIGS. 28 and 30. Leakage is most likely to occur at these points ofmaximum gauge pressure, with positive pressure tending to expel fluidout of the pathway and negative pressure tending to draw fluid into thepathway from a neighboring well or channel. (This assumes that theneighboring channels and wells are held at zero gauge pressure when notsubjected to flow. Another method to control the pressure differentialsacross the system walls is to apply matching pressures to theneighboring pathways while they are not under flow.) At intermediatepoints along the fluid pathway, the pressure differential across thesystem walls has values between ±ΔP and zero for positive and negativelypumped systems, respectively.

The same end-to-end pressure differential ΔP is exerted across thepathway by establishing pressures ±ΔP/2 at the input and outputreservoirs, respectively. In this case, the maximum gauge pressuresexerted across the walls of the system are ±ΔP/2 and occur at thecorresponding endpoints of the system. Moreover, the applied gaugepressure within the device is close to zero if the wells are roughly atthe midpoint between the input and output reservoirs along the length ofthe pathway. At the endpoints of the pathway, the maximum pressuredifferences ±ΔP/2 are more readily held without leakage within plastictubing or silica capillaries. Detection of leakage is facilitatedbecause it is likely to occur only at the system endpoints.

In one embodiment, the two syringes of a dual pumping system are thesame diameter and volume, and because they are linked to the sameactuator in a push-pull configuration, each syringe is moved the samelinear distance, and identical volumes are exchanged for each incrementof movement. As any particular source compound is pushed into the array,an equal volume of waste fluid is pulled out. Thus, the array of msource syringes also contains an array of m negative pressure wastereservoirs. This array of negative pressure waste reservoirs ismultiplexed to the column channels by a 1×m valve-less manifold, just asthe array of source reservoirs is multiplexed to the row columns.

In another embodiment, the source and waste syringes are of differentdiameter, and the syringes must be controlled by two separate driversthat drive each syringe at a different linear rate, but yield the samevolume changes in each syringe. For example, a smaller diameter syringecould be used for a compound, while a larger diameter syringe could beused as the waste receptacle. If the diameter ratio is a factor of 2, asthe driver of the small syringe moves one linear unit to expel fluid,the driver of the large syringe moves only 1/4 linear unit to withdrawfluid, but the volumes exchanged are identical. FIG. 31 shows oneparticular case of this embodiment of a “dual-pumped” system, in whichthere is only one waste syringe pump that must be actuated incoordinated fashion (e.g., by means of computer control) with theactuation of any particular source syringe pump. Alternatively, thenegative pressure pump may be a capillary action pump, also shown inFIG. 31. For the capillary action pump, the “actuation” of the pump isachieved by means of valves.

In summary, the dual-pumped system has the following advantages:

1) reduction in the maximum applied gauge pressure,

2) the applied gauge pressure is small near the midpoint of the pathway,and

3) leakage is readily detected and most likely to occur at the systemendpoints.

Higher-Level Multiplexing

The use of multi-port flow manifolds between the pumped reservoirs andthe crossed channel array allows m different pumps (200) or reservoirs(220) in these various embodiments, with m different liquid or vapormixtures to selectively and sequentially produce flow in each well (j,k)of the n×n well array (002). More generally, a set of wells defining asingle fluid pathway {(j,k), (j,n), (m,k), (m,n) . . . etc.} can bedefined by this system. Flow of only one liquid or vapor mixture canoccur through only one fluid pathway at a time. Higher levelmultiplexing, allowing simultaneous flow of different liquid or vapormixtures through different pathways, is possible with a more complexsystem of flow manifolds. Such a system is easily constructed as ageneralization of the present design.

Control of Diffusion

The principle of this embodiment is that the valve element (520) and thevalve seat (540) are shaped to allow flow in one direction and tosuppress diffusion when flow is stopped.

Furthermore, the restoring forces on all valves are applied “passively”and constantly. No detailed control system for the check valves isneeded, because the control of fluidic pressure gradients is the meansby which individual valves are opened.

The use of crossed microchannels with a large number of open pathwaysbetween, for example, the 2n microchannels and the n² wells, may in someembodiments of the invention be augmented by additional methods tocontrol the diffusion of compounds between wells and microchannels.Unlike prior art systems, the microfluidic system of the presentinvention does not need a detailed control system within the device forcontrol of well-to-well diffusion. Two exemplary means of controllinginter-well diffusion are disclosed that do not require the activecontrol of on-board valves. Both of these methods block inter-welldiffusion by means of fluid flow control from valves, pumps, andmanifolds that are external to the array of wells, and thus conform tothe overall design principles of the invention: the optimal imaging ofsub-arrays of wells in a class of fluidic architectures that requiresfewer channels and permits smaller inter-well spacings.

The first method uses the insertion of segments of air between differentfluid segments. The positive and/or negative pressure methods given inthe above embodiments are employed to insert these air segments. Ingeneral, the application of differential pressure in appropriatesequences across a particular rows {j} and columns {k} or {k*} can placea number of separate air segments in the input channels {j} between anumber of different fluids. In particular, appropriate sequencing candirect one fluidic segment to one well (j,k) and direct another fluidicsegment to another well (j, k−1), while also inserting and maintainingan air segment in row j between these two wells. Subsequently, the airsegment can be passed out of the system through an unused well or via arinse channel k* (130)(FIG. 35).

The second method of diffusion control uses “normally-closed” checkvalves (500) between every pair of adjacent vias (via k and via k+1)(180) that connect the channels j to the wells (j, k) and (j, k+1) (FIG.36). Similarly, check valves may be used in architectures incorporatingrinse column channels {k*}. In other embodiments, check valves may beplaced within the vias (160, 180) that connect wells to microchannels orrow channels to rinse column channels. These valves are closed unless apressure gradient is applied across a fluid pathway of the array. Thenonly those valves distributed along the selected fluid pathway arepushed open. When flow is stopped and the valves return to the closedposition, the valves suppress diffusion of compounds between wells andmicrochannels, or row channels and rinse channels. The restoring forcethat closes each valve element (520) in its corresponding valve seat(540) may be provided by any number of methods known in the art. Thestrength of the restoring force must allow the valves to open upon theapplication of pressure gradients that yield appropriate flow rates(typically less than 1 cm/min.). Methods for application of restoringforces include mechanical force by the deformation of metallic,semiconductor, or organic materials; pneumatic pressure; electrostaticforce, magnetic force, and the force of gravity.

In another embodiment, an externally-applied magnetic field gradient(620) induces a restoring force on valve elements (520) that incorporatea magnetic material. This includes but is not limited to the use ofparamagnetic beads (600) (e.g., supplied by Dynal Corp.) as shown inFIG. 37. These paramagnetic beads (600) serve as balls in micro ballvalves that are fabricated within and along each row and column channel.U.S. Pat. Nos. 5,643,738 and 5,681,484 describe a magnetically-actuatedmicroball check valve. However, these patents particularly describe anactively-controlled valve that would permit closure of the ball valve“on demand,” and has as its purpose to stop flow that is imposed by someexternal pressure. In the instant case, the particular advantage to themicrofluidic architectural system is that a mild restoring force issimultaneously applied to a large ensemble of ball valves throughout thearray, and no active control is required. The movement of selected balls(whenever it is desired to open the valves) along a fluid pathway isachieved by means of causing the fluid itself to flow. The ball valve iscontrolled by the flow of fluid, not the flow of fluid is controlled bythe ball valve. Moreover, in this embodiment, the valve seat isparticularly designed so that a seal is not obtained in one direction,as opposed to the other direction, where a mild seal sufficient to stopdiffusion is obtained.

In one embodiment, one micro ball valve is located between every pair ofadjacent vias (via k and via k+1) that connect the channels j to thewells (j, k) and (j, k+1). Each micro ball valve opens only when apressure gradient is applied across a pathway that includes that valve.The design of the valve seat (540) on one side is shaped (e.g., withgrooves) so that flow is not stopped when fluidic pressure moves theball in the direction of flow. When no pressure gradient is applied (andno fluid is flowing) at the site of a particular ball valve, a magneticrestoring force presses that paramagnetic ball back against the otherside of the valve seat (540)(where the seat is shaped to fit the ball)to suppress diffusion of compounds across that portion of themicrochannel. This restoring force is induced by a magnetic fieldgradient applied to the entire array, so that all valves are normallyclosed.

In another embodiment, an externally-applied magnetic field gradient(620) induces a restoring force on valve elements (520) that incorporatea ferromagnetic material. For example precision chrome steel balls areavailable from Glenn Mills Inc., Clifton, N.J. in a range of sub-mm tofew mm diameters.

FIG. 38 shows an embodiment of a shaped valve seat that exemplifies thisdesign principle. Specifically, diffusion past the ball is stopped whenthe externally-applied restoring force presses the ball to the left intoa round opening in the valve seat. When flow is actuated byexternally-applied pressures, the ball is forced to the right, but theball does not stop the flow because the right side of the valve seat isoval shaped and includes a groove-like deformation at along one side,forming path for fluid to bypass the ball in this open position of thevalve.

FIG. 39 shows an embodiment that allows a single magnetic field gradientapplied to the whole cassette of the invention to induce force on both aset of ball valves of the input channels and a set of ball valves of theoutput channels

In one embodiment the direction of the magnetic field gradient isparallel to the plane of the array, but oriented at 45 degrees to boththe row and column channels. Thus each ball experiences a component offorce in the direction of the “closed” side of its corresponding valveseat (540). In other embodiments, the magnetic field gradient is appliedperpendicular to the plane of the array, and the ball valves operate inthe vertical direction within the horizontal microchannels or within thevertical vias. In the latter case (vertically-actuated ball valveswithin vertical vias) the via architecture must again allow each ball tobe subjected to a component of magnetic force in the direction of the“closed” side of its corresponding valve seat (540).

An example has been given of a ferromagnetic or paramagnetic valveelement (520) of spherical shape, but other shapes and other restoringforces may generally be used that function in the same manner.

Well Dimensions and Characteristics

In another aspect, the present invention provides a cassette for cellscreening, comprising a substrate having a surface, wherein the surfacecontains a plurality of cell binding locations; a fluid delivery systemfor delivering reagents to the plurality of cell binding locations,wherein the fluid delivery system comprises a multi-level chamber thatmates with the substrate, wherein the multi-level chamber comprises acrossed array of microfluidic input channels and output channels and aplurality of fluidic locations in fluid connection with the microfluidicinput channels and output channels; and a plurality of wells, wherein anindividual well comprises the space defined by the mating of one cellbinding location and one fluidic location, and wherein the wells arepresent at a density of at least about 20 wells per square centimeter.In a preferred embodiment, the well density is between about 20 wellsper square centimeter and about 6400 wells per square centimeter.

In a preferred embodiment, the cassette of this aspect of the inventionfurther comprises one or more input manifolds in fluid connection withthe microfluidic input channels one or more output manifolds in fluidconnection with the microfluidic output channels. In another preferredembodiment, the cassette of this aspect of the invention furthercomprises at least one source receptacle in fluid connection with theone or more input manifolds and at least one waste receptacle in fluidconnection with one or more output manifolds. The various controldevices in these embodiments are as described above.

No limitation is placed on the well shape geometry in the instantinvention, as the well shape may differ in different embodiments of theinvention. While a rectangular well shape allows for the minimumdistance and area between wells in the array, this shape has thedisadvantage of potential differences in cell culture or fluidicproperties in the corner regions of the wells. Therefore, a well shapethat is circular or that is rectangular with rounded corners is used ina preferred embodiment of the invention.

The array system of the present invention is designed for the imaging ofindividual cells within a field of cells in a drug screening assaysystem. Screening with imaging assays that involve the measurement ofeach individual cell within a field of cells, in parallel, is referredto as High Content Screening (HCS), because detailed information aboutintracellular processes is contained within the image of a single cell.Screening with lower resolution imaging or with detectors that integratea signal over a population of cells is referred to as High ThroughputScreening (HTS), because it is faster. In HCS, the cells are typicallyidentified by imaging the nuclei of the cells, which are spherical orellipsoidal in shape with a long-axis length that ranges typically from5 μm to 15 μm. The cell array used in the present invention optimizesthe rate of HCS by using a higher-density multi-well format compared toconventional 96-well plates.

For HCS, the number of cells that provide a statistically-relevantsample as well as the number of cells that are required per unit surfacearea varies depending on the type of assay and the type of cell beingcultured. Thus, the minimum size of the well required by statisticalcriteria for each assay is different. Nevertheless, no matter what thatminimum or optimum well size is, the rate at which wells can be readwith adequate image resolution to resolve individual cell nuclei isoptimized by the present device, which allows several wells to be imagedat one time in parallel. This is done most efficiently if there isminimal wasted space between wells, and the cell-plated area within theentire image is therefore maximized.

Moreover, the present array system optimizes the flexible implementationof both HTS and HCS on the same optical imaging cell-based screeningsystem. The required pixel density (pixels per micron) is lower for HTS(for example, 0.001 to 0.1 pixels per micron) and higher for HCS (forexample, 0.1 to 4.0 or more pixels per micron). The present systemprovides seamless, combined use of HTS and HCS, where a “hit” identifiedin HTS is also read in HCS mode for more detailed analysis. In fact, theavailability of HCS on the same platform and with the same sample,greatly increases the information associated with “hits” identified inthe HTS mode. Nevertheless, the fastest possible rate of HCS isadvantageous to allow maximum overall screening throughput whenever HCSis used. The minimum pixel density required for HCS in combination withthe maximum well density of the array are the two critical parametersthat define the maximum rate at which wells can be read in the HCS mode.This invention describes how the design of the present array system canmaximize this HCS rate, as well as the HTS rate.

In use with 96-well microplates, a conventional microscope imagingsystem can image square fields that are 0.1 mm to 10 mm wide centered oneach 7-mm diameter well. The wells are imaged or “read” in series. Ateach position of the stage, typically, only one area of a well is read.To read the entire array (either in HTS or HCS modes) the sample stagemust move the microplateat least 96 times.

In this invention, we define a novel cell screening method in which manywells are read at one time. Because the present array has well widthsand inter-well distances that are roughly 10 times smaller than in the96-well microplate (wells that are sub-millimeter in width and overallplate dimensions of several millimeters rather than several centimeterswide), a cell screening system, such as that disclosed in the presentinvention, can image many wells simultaneously. Thus, the sample stageis moved fewer times per plate and several wells are read in parallel(FIG. 39).

This essentially involves the serial imaging of “sub-arrays” (020) ofthe entire microwell array (040), while acquiring data from all wells ofeach sub-array in parallel. For example, the imaging of 3×3 wellsub-arrays requires 9 times fewer movements of the sample stage, andallows a roughly 9-times faster rate of data acquisition.

For a given CCD detector array (080), the magnification of the opticalsystem (060) determines the area of the sub-array (020) that isprojected onto the detector. For example, an image pixel density of 1pixel per micron is achieved for a 1000 μm×1000 μm sub-array of wells byusing 10× optical magnification and a 1000×1000 pixel CCD array of 10μm×10 μm elements. In a second example, a 2000 μm×2000 μm sub-array ofwells can be imaged onto a 2000×2000 pixel CCD array at the samemagnification to yield the same image pixel density. In a third example,an image pixel density of 0.1 pixel per micron is achieved for a 10mm×10 mm sub-array of wells by using 1.0× optical magnification and a1000×1000 pixel CCD array of 10 μm×10 μm elements. In a fourth example,a 10 mm×10 mm sub-array of wells can be imaged onto a 2000×2000 pixelCCD array at 2.0× magnification to yield the same image pixel density.

Below we give embodiments of particular embodiments of well sizes andspacings according to the present invention that optimize the imaging ofsub-arrays. These well sizes must be large enough to contain a desirednumber of cells bound to a desired number of cell binding sites withineach location of the array. Due to the considerable range of desiredcell densities to be cultured on the cell binding sites (i.e.: culturesof very widely spaced cells, or of confluent monolayers may be desired),the desired well size may range from that which contains only one cellon a very small cell binding site of about 10 μm in diameter (i.e.: awell size of 20 to 50 microns), to that which contains either one largecell binding site or an array of many cell binding sites (i.e.: a wellsize of 1 to 2 mm) comprising a single location of the array. The formercase leads to higher well densities, and the latter to lower welldensities.

For both high and low well densities, and for wells that contain one ormany cell binding sites, the class of microfluidic architecturesaccording to the present invention permit the optimal spacing of wells,the minimal wasted space between the wells, and the maximum number ofwells that can be simultaneously imaged in a sub-array and still obtainadequate pixel resolution in the image.

We now use the four examples of optical resolution given above toillustrate how for a preferred range of sub-array sizes, opticalmagnifications, and pixel resolutions, and based on the advantages ofthe microfluidic architecture described herein, the present devicesupports well sizes and spacings that enable great increases in thespeed at which the wells can be imaged or read. Additional sub-arraysizes, optical magnifications, and pixel resolutions are supported bythe methods and devices of the invention. The scope of the invention isnot limited by the current state of the art in the number of availablepixels, nor in the pixel sizes in electronic imaging systems.

-   1. For the first example above, a 1000 μm×1000 μm sub-array of wells    is imaged onto a 1000×1000 pixel CCD camera (with image resolution    of 1 pixel per micron). We now give four examples of well sizes and    densities that are supported by this invention. (a) First, a 2×2    sub-array of 300 μm×300 μm wells with 200 μm-thick walls and a well    density of 400 wells per cm². Imaging these wells in groups of 4    yields a 4-fold speed increase compared to reading the plate one    well at a time. (b) Second, a 3×3 subarray of 200 μm×200 μm wells    with 100 μm walls yields a 9-fold increase in speed and a well    density of 1111 wells per cm². The number of wells per unit area is    a factor of 80 greater than that provided by the current highest    density commercial microplate (the 1536 well plate). (c) Third, a    5×5 sub-array of 100 μm×100 μm wells and 100 μm walls yields a    25-fold speed increase and a density of 2500 wells per cm². (d)    Fourth, for an even higher density of wells and a greater speed    advantage, the well can be 25 μm×25 μm with 100 μm walls, yielding    an 8×8 sub-array, a 64-fold speed increase, and a well density of    6400 cells per cm².-   2. For a 2000 μm×2000 μm sub-array of wells imaged onto a 2000×2000    pixel CCD camera (with image resolution of 1 pixel per micron), one    additional example of well density is considered in addition to the    four examples considered above. (a) First, a 2×2 sub-array of 800    μm×800 μm wells with 200 μm-thick walls yields a 4-fold speed    increase and a well density of 100 wells per cm². The number of    wells per unit area is a factor of 5 greater than that provided by    the current highest density commercial microplate (the 1536 well    plate). (b) Second, a 4×4 sub-array of 300 μm×300 μm wells with 200    μm-thick walls yields a 16-fold speed increase and a well density of    400 wells per cm². (c) Third, a 6×6 sub-array of 200 μm×200 μm wells    with 100 μm walls yields a 36-fold speed increase and a well density    of 900 wells per cm². (d) Fourth, a 10×10 sub-array of 100 μm×100 μm    wells with 100 μm walls yields a 100-fold speed increase and a well    density of 2500 wells per cm². (e) Fifth, a 16×16 sub-array of 25    μM×25 μM wells with 100 μm walls yields a 256-fold speed increase    and a well density of 6400 wells per cm^(.)-   3. For a 10 mm×10 mm sub-array of wells imaged onto a 1000×1000    pixel CCD camera (with image resolution of 0.1 pixel per micron), we    again describe the four cases of well sizes and densities described    in example 1 above. (a) First, a 20×20 sub-array of 300 μm×300 μm    wells with 200 μm-thick walls yields a 400-fold speed increase and a    density of 400 wells per cm². (b) Second, a 30×30 sub-array of 200    μm×200 μm wells with 100 μm walls yields a 900-fold speed increase    and a well density of 1111 wells per cm². As before, this case    supports a well density 80 times greater than that provided by the    current highest density commercial microplate. (c) Third, a 50×50    sub-array of 100 μm×100 μm wells with 100 μm walls yields a    2500-fold speed increase and a well density of 2500 wells per    cm². (d) Fourth, an 80×80 sub-array of 25 μm×25 μm wells with 100 μm    walls yields a 6400-fold speed increase and a density of 6400 wells    per cm². For an even higher density of wells and a greater (2500    fold) speed advantage, the wells can be 100 μm×100 μm with walls    separating the wells that are 100 μm wide, yielding 2500 wells per    sub-array, and 2500 wells per cm².

4. For a 10 mm×10 mm sub-array of wells imaged onto a 2000×2000 pixelCCD camera (with image resolution of 0.1 pixel per micron), the casesfrom example 2 above of 100, 400, 1111, 2500, and 6400 wells cm² againapply, but yield 100 times greater speed increases because the subarraysare 10 times wider on each side. Thus, the speed increases for welldensities of 100, 400, 1111, 2500, and 6400 wells/cm2 are 400×, 1600×,3600×, 10,000×, and 25,600×, respectively, in the 10 mm×10 mm array.TABLE 2 The following table compares well pitches for various otherdevices: Well to well Estimated Speed Type of plate Reference distance(mm) Wells/cm² increase* Standard 1536 well plate 2.25 20 12 × 12 arrayin 1.5″ × 1.5″ Gen. Engr. News 18:12 3 11 area (1998) 864 well plate: 24× 26 U.S. Pat. No. 5,910,287 3 11 array in std. microplate footprint of108 cm² 9600 well plate: 80 × 120 U.S. Pat. No. 5,910,287 0.9 123 arrayin a std. microplate footprint of 108 cm² Present invention 1 mm × 1 mmsubarray: Example 1(a) 0.5 400 4X 1000 × 1000 pixel CCD Example 1(b) 0.31111 9X Example 1(c) 0.2 2500 25X Example 1(d) 0.125 6400 64X 2 mm × 2mm subarray: Example 2(a) 1 100 4X 2000 × 2000 pixel CCD Example 2(b)0.5 400 16X Example 2(c) 0.3 1111 36X Example 2(d) 0.2 2500 100X Example2(e) 0.125 6400 256X 10 mm × 10 mm subarray: Example 3(a) 0.5 400 400X1000 × 1000 pixel CCD Example 3(b) 0.3 1111 900X Example 3(c) 0.2 25002500X Example 3(d) 0.125 6400 6400X 10 mm × 10 mm subarray: Example 4(a)1 100 400X 2000 × 2000 pixel CCD Example 4(b) 0.5 400 1600X Example 4(c)0.3 1111 3600X Example 4(d) 0.2 2500 10,000X Example 4(e) 0.125 640025,600X*Estimated speed increase is a comparison of the speed at which theentire array could be imaged by sub-arrays compared to imaging byseparately imaging each well

All of these particular aspects of the instant microfluidic device—thecrossed-channel, multilevel architectures, the high well density, thefluid flow control into these architectures, the arrangement of wellsinto spatially-optimized sub-arrays of wells, and the optional means ofcontrol of inter-well diffusion of compounds—are specifically selectedand designed to form an integrated system that is compatible with theuse of cell culture medium (a polar, aqueous solution containingbiological macromolecules and salts), with the maintenance of desired,physiological levels of dissolved oxygen and carbon dioxide gases in themedium, and with the maintenance of desired, physiological temperature(typically 37° C., but other temperatures in the approximate range of15° C. to 40° C. may be desired for particular cell types).

Particularly, the cell culture medium may be equilibrated in off-boardvessels to the desired levels of temperature and dissolved carbondioxide and oxygen. Then, using off-board vessels and valves, the mediumis moved through the microchannels and to the wells. The sealing ofthese microchannels and wells from the atmosphere enables the partialpressures of gasses to be controlled by means of the equilibration ofthe external vessels. In a preferred embodiment, the level of carbondioxide in the media within the wells may be established and maintainedby equilibrating the media prior to its flow into the device, and/or byallowing exchange of a mixture of carbon dioxide and air with the mediaas it sits within the well. A number of different environmentallycontrolled chambers for live cell culture exist. (U.S. Pat. Nos.5,552,321 and 4,974,952; Payne et al., J. Microscopy 147:329-335 (1987);Boltz et al., Cytometry 17:128-134 (1994); Moores et al., Proc. Natl.Acad. Sci. 93:443-446 (1996); Nature Biotech. 14:3621-362 (1996)).

In a further preferred embodiment, the temperature of the entiresubstrate is controlled by being in contact with, or composed integrallywith, a heater and with a temperature sensor. An electronic temperaturecontrol system regulates the heater to attain a chosen temperature setpoint. Thus, the design of the microfluidic array system described inthis invention is readily used in a way that supports live cell culturewithout an external incubator system. FIG. 12 shows how this inventionprovides for the automated reading of cassettes. The cassettes are keptunder controlled environmental conditions within two storagecompartments (48 and 54) before and after reading in the luminescencereader instrument 44. While being read by the luminescence readerinstrument, the system maintains the appropriate temperature andcomposition of dissolved gasses in the cell culture media within thewells of the cassette. Temperature control within the wells is providedby heating device(s) and temperature sensor(s) within the cassette ofthe luminescence reader instrument. Control of dissolved gas compositionwithin the wells is provided by equilibration of the media with a sourceof pre-mixed air and carbon dioxide (available commercially) prior tothe flow of media into the wells of the cassette. Any type of controllerfor regulating gas partial pressures may be used with the instantinvention. In a preferred embodiment, the system comprises a gascontroller comprising a pre-mixed gas source connected to a reservoir orreservoirs containing cell culture media and/or test compounds. A gaspressure regulator and flow control valve control the rate of flow ofthis gas mixture and its pressure in the connected fluid reservoir(s).This equilibration of the gas mixture with the fluids may be carried outin reservoirs either on-board or off-board the cassette, but in anycase, outside the matrix of the wells.

The present invention fulfills the need in the art for devices andmethods that decrease the amount of time necessary to conduct highthroughput and/or high content cell-based screening. The devices of theinvention are also ideally suited as a cell support system for ahand-held diagnostic device (i.e.: a miniaturized imaging cell-basedassay system). The drug discovery industry already uses 96-wellmicroplates and is in transition towards the use of 384-well plates.Plates with up to 1536 wells are envisioned. Thus, there is a greatadvantage in both throughput and economy from the use of even higherdensity plates, such as that of the present invention.

The sealed containment of the cells in the cell array of the instantinvention will provide a rugged system that is portable and usable inany orientation. For military and civilian toxin testing, the devices ofthe instant invention will provide two primary advantages. First, toxintesting based on cellular function is advantageous compared to toxintesting based on molecular structure (e.g., mass spectrometry or opticalspectroscopies), because it tests the ultimate effect of the compound ontissue rather than a related property of the compound whose link totoxicity may be unknown or dependent on other conditions. Second, anautomated, miniaturized, sturdy, and portable format forcellular-function based sensor would be advantageous compared to thecurrent state-of-the-art that involves large microplates filled by largerobotic pipetting systems and read by large microscopic readers.

Additionally, other assay capabilities can be integrated into the deviceof the present invention that are not possible in prior art devices,such as simple plastic microplates. For example, a mass spectrometric orcapillary electrophoretic analytical device, or systems for DNA and/orprotein analysis, can be integrated in the instant device to measurechemical and structural parameters of test compounds. Such integrationwould provide information that is complementary to the cell-based,functional parameters measured by a HCS or HTS system. For conventionalmicroplates, such additional assay functions are external to the plate,and therefore require extensive additional equipment for both thetransfer of samples from the microplate and for the analytical equipmentitself. In the miniaturized format with integrated fluidics of thepresent invention, the sample is integrally pumped to a microanalyticalsystem on the chip or on an integrally-connected second chip. Suchmicroanalytical chips will reduce the cost and dramatically increase thespeed of cell-based analysis. In this context, the marriage of amicroscale live-cell analytical system with a microscale chemicalanalytical system is expected to provide great improvements overexisting methods and devices.

EXAMPLE 4 Cell Patterning

Polymeric and glass surfaces in their native structures have been usedas cellular growth substrates for decades. Differing techniques havebeen utilized to adjust the surface chemistry of these materials to makethem more attractive for cell adhesion, including adsorption of celladhesion molecules; sulfonisation of the material (European PatentApplication 576184); co-polymer blends of extracellular matrix proteinfragments such as RGD (U.S. Pat. No. 5,733,538); and chemical oxidation(using solution chemistry) of the surface for further chemicalmodification (using solution chemistry) (U.S. Pat. No. 5,330,911) suchas silanes (U.S. Pat. No. 5,077,085) or thiols (U.S. Pat. No.5,776,748).

In addition to adjusting the surface of these substrates to render themmore attractive for cellular adhesion, techniques have been developed torender the surfaces repulsive for cellular adhesion. Cell repulsivesurfaces have been achieved by utilizing extremely hydrophobic surfaces(contact angle >90°), which show cell repulsion for several days.(Dulcey et al., Science 252:551 (1991)). A more widely utilized methodemploys extremely hydrophilic surfaces (contact angle approximately 0°),by immobilizing sugars and oxygen-rich moieties to the surface throughcommon linking chemistries. These surfaces demonstrate longer periods ofcell repulsion before degrading and allowing cell adhesion. The mostutilized molecule for cell repulsion is oxygen-rich poly(ethyleneglycol) (PEG). PEG can be attached to polymeric and glass substrates bymethods including chemically activating the substrate to react with apoly(ethylene imide)-PEG molecule (Brink, Colloids and Surfaces, 1992.66: p. 149-156), aminating an activated surface and reacting it withbifunctional electrophilic molecules such as PEG-epoxide (Bergstrom, K.,Journal of Biomedical Materials Research, 1992. 26: p. 779-790; WO98/32466 EP0576184, Nilsson, D., in Methods in Enzymology, 1984. 104: p.56-69; Sofia, S., Macromolecules, 1998. 31: p. 5059-5070), andphotochemically through photolabile PEG-conjugates such asPEG-benzophenone and PEG-styrene co-polymer blends. (Becker,Makromolecular Chemistry, 1982. 3: p. 217-223; U.S. Pat. No. 5,502,107)

The techniques mentioned so far will lead to homo-monolayers, containingone of the cell attractive or cell repulsive moieties. A combination ofthe above technologies can lead to the creation of hetero-monolayers. Ifthe positioning of these cell adhesive and cell repulsive cues can becontrolled to a high degree, cells can be patterned on the substrate ofchoice. Cell patterning has been achieved on glass and metalized glasssubstrates utilizing silanes (U.S. Pat. No. 5,077,085), thiols (U.S.Pat. No. 5,776,748), and azidos (U.S. Pat. No. 5,593,814). These methodsprovide selective localization of cells using a multi-step, equipmentintensive process, and/or irreproducible techniques such as deepultraviolet ablation of molecules, and/or printing by mechanicalstamping, and/or require a polymer layer tens of nanometers (nm) inthickness, which changes the optical quality of the substrate.

Thus, there is a need in the art for affordable, facile, equipmentinsensitive, reproducible methods for cell patterning on durablesubstrates such as glass and plastic that do not decrease the opticalquality of the substrate, as well as for the cell patterning substratesthemselves. Most surface modification processes for glass and siliconwafers are not amenable to plastic due to the nature of the harshsolvents used. Thiols are unsuitable for coating on plastics as theyrequire a coinage metal for forming a coordination bond with thesubstrate. Silanes, though amenable to coating on plastics, require ahydroxylated surface, such as presented by glass and silicon, to form acovalent bond with the substrate.

Thus, in one aspect, the present invention provides novel methods formaking a substrate for selective cell patterning. In one embodiment, themethod comprises

a) providing a substrate with a surface, wherein the surface containsreactive hydroxyl groups;

b) contacting the hydroxyl groups on the surface of the substrate with abifunctional molecule comprising a hydroxy-reactive moiety and anucleophilic moiety to form a monolayer;

c) applying a stencil to the substrate;

d) applying an electrophilic cell repulsive moiety to exposed regions ofthe monolayer to form a covalent bond between the cell repulsive moietyand the bifunctional nucleophile deposited in step b to form cellrepulsive locations;

e) removing the stencil; and

f) applying cell adhesive molecules to the substrate to produce cellbinding locations, wherein the cell adhesion molecules bind thesubstrate only in positions that were contacted by the stencil.

In another embodiment, the method comprises

a) providing a substrate with a surface, wherein the surface comprisesreactive hydroxyl groups on the surface of the substrate;

b) contacting the hydroxyl groups on the surface of the substrate with abifunctional molecule comprising a hydroxy-reactive moiety and anucleophilic moiety to form a monolayer

c) applying a stencil to the substrate;

d) applying cell adhesive molecules to exposed regions on the monolayerto produce cell binding locations;

e) removing the stencil; and

f) applying an electrophilic cell repulsive moiety to the substrate toproduce cell repulsive locations, wherein the cell repulsive moietyforms a covalent bond with the bifunctional nucleophile deposited instep b only in positions that were contacted by the stencil.

The surface reactive hydroxyl groups can either be naturally occurring,or can be introduced via any technique known in the art. For example,polymer or glass can be oxidized so that they present surface hydroxylgroups, which react with organosilanes to produce covalentSi—O-substrate (siloxane) linkages. (U.S. Pat. No. 5,077,085)

In a preferred embodiment, oxidation is accomplished by oxygen plasmatreatment, which can be achieved using oxygen doped radio frequency glowdischarge. This discharge is accomplished with an instrument that canproduce charged particles (electrons and positive ions) that interactwith the background gas (oxygen), to produce free radicals under thetime-varying electric field in radio frequency. The sample is placedinto a cylindrical reactor, a minimal amount of oxygen gas isintroduced, and charged particles are evolved between parallel-platedelectrodes resulting in the cleavage of the O₂ bond. After thiscleavage, high-energy free radicals can insert themselves into thepolymer backbone resulting in the formation of various oxygen moieties,among which are hydroxyl groups. (U.S. Pat. No. 5,357,005; U.S. Pat. No.5,132,108)

As used herein, the bifunctional molecule comprises

(a) a hydroxyl-reactive electrophile, including but not limited tosilanes, carboxymethyl groups, succinimides, succinimidyl succinates,benzotriazole carbonates, glycidyl ethers (or epoxides),oxycarbonylimidazoles, p-nitrophenylcarbonates, aldehydes, isocyanates,and tresylates; and

(b) a nucleophile, including but not limited to sulfhydryl groups, aminegroups, hydroxyl groups, or proteins or fragments thereof, peptides, andsynthetic ligands for cell surface receptors, wherein the nucleophilecan bind to other molecules and/or cells.

In one embodiment, the bifunctional molecule comprises an organosilane,wherein silane is the electrophile, and the nucleophile includes, but isnot limited to sulfhydryl groups, amine groups, hydroxyl groups, orproteins, peptides, and synthetic ligands for cell surface receptors,wherein the nucleophile can bind to other molecules and/or cells. Asused herein, organosilanes fall into a larger class of molecules, whichhave the capability of forming self-assembled (SA) films. The generalform of this molecule comprises R_(n)SiX_(4-n), where n=1, 2, or 3;X═Cl, OCH₃, or OC₂H₅, and R is the nucleophile as described above.

In a preferred embodiment, the bifunctional molecule comprises anaminosilane, wherein silane is the electrophile that attaches to thesurface hydroxyl groups, and an amine group is the nucleophile that canbind to other molecules and/or cells.

In a more preferred embodiment, the aminosilane is selected from thegroup consisting of methoxy or ethoxy silanes, which include but are notlimited to trimethoxysilylpropyldiethylenetriamine,trimethoxysilylethylenediamine, aminopropyltriethoxysilane,trimethoxyaminopropylsilane, or chlorosilanes such astrichlorosilylethylenediamine, aminopropyltrichlorosilane. In a mostpreferred embodiment, the amino silane istrimethoxysilylpropyldiethylenetriamine

As used herein, a cell adhesive molecule includes compounds that: (1)introduce charge; and/or (2) are polar; and/or (3) contain sulfur and/oramines; and/or (4) are capable of tethering cells or other cell bindingmoieties, such as proteins, peptides, and synthetic ligands for cellsurface receptors, thereby creating a cell binding location.

As used herein, the term cell repulsive moiety includes compounds thatare capable of directly inhibiting cell binding, or that bind to othermoieties which inhibit cell binding to the location, includingpolyethylene glycol (PEG) and other oxygen-rich compounds, sugars,hydrogels, extremely hydrophilic surfaces, or extremely hydrophobicsurfaces.

In all of these embodiments, the cell adhesive molecule and/or cellrepulsive moiety can be applied to the substrate via solution or vaporphase deposition. In a preferred embodiment, vapor deposition of thecell adhesive molecule and/or cell repulsive moiety is utilized. Forexample, apor phase deposition of various silanes has been demonstrated.(Tripp et al., Langmuir 8:1120-1126 (1992); Moses et al., AnalyticalChemistry 20:4 (1978) In most cases, rather than adding the sample to asolution of silane, a hydroxylated surface is placed in the presence ofvaporized silane (achievable by traditional vacuum techniques). Thereaction takes place at the surface and results in self-assembledmonolayers similar to that of silane solution deposition.

A wider range of cell adhesive molecules and cell repulsive moieties canbe used with vapor phase deposition, because a solvent is not needed.For example, many silane solvents would dissolve the polymeric substrateand destroy its optical quality. In this embodiment, the methodcircumvents the use of solvents altogether.

In another preferred embodiment, the cell repulsive moiety comprises anamine-reactive moiety, including but not limited to2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride)-activatedpolyethylene glycol (PEG), polyvinylpyrrolidone, polyvinylalcohol, orany other amine-reactive extremely hydrophilic compound such as sugars(mannitol) or PEG, where the amine-reactive part can include, but is notlimited to, carboxymethyl groups, succinimides, succinimidyl succinates,benzotriazole carbonates, glycidyl ethers (or epoxides),oxycarbonylimidazoles, p-nitrophenylcarbonates, aldehydes, isocyanates,and tresylates; or any amine-reactive extremely hydrophobic compoundsuch as tridecafluoro-1,1,2,2-tetrahydrooctyl groups (13f) where theamine-reactive part can include, but is not limited to carboxymethylgroups, succinimides, succinimydyl succinates, benzotriazole carbonates,glycidyl ethers (or epoxides), oxycarbonylimidazoles,p-nitrophenylcarbonates, aldehydes, isocyanates, and tresylates. In amost preferred embodiment, the amine-reactive cell repulsive moietycomprises tresyl chloride-activated polyethylene glycol(“tresyl-chloride activated-PEG”).

The chemistry of the tresyl-activated PEG can be used to regulatesurface hydroxyl, amine, or sulfhydryl groups. Tresyl chloride willallow stable linkages to be formed between the nucleophile and theinitial hydroxyl, amine, or sulfhydryl group carrying carbon. In apreferred embodiment, PEG is attached to a tresyl group for reactionwith surface aminosilane groups.

In these preferred embodiments, cell adhesive cues can be defined by theuse of a stencil, which has no size constraints. Cell repulsive cues,which also can be defined by the stencil, are tethered to an aminosilanemonolayer. The cell binding locations may optionally be coated with celladhesive proteins, protein fragments, or peptides, and seeded with cellsresulting in a patterned array of cells.

This hydroxylated substrate is contacted with a bifunctional moleculecomprising an electrophile and a nucleophile. This modified substrate iscontacted with a textured elastomeric substrate (herein referred to as a‘stencil’), such as rubber, polyurethanes and poly(dimethyl) siloxanes(“PDMS”), to form a hermetic seal between defined regions of the stenciland the modified substrate. In a preferred embodiment, the stencilcomprises PDMS. These materials are quite affordable, providing asignificant benefit over traditional UV photolithography methods thatemploy a costly, high energy laser apparatus. (U.S. Pat. No. 5,077,085)

The stencil comprises a physical mask that enables physical protectionof defined regions of the underlying substrate from the subsequentsolution or vapor phase deposition of the cell repulsive or celladhesive moiety. This disclosed method of using a ‘physical mask’distinguishes itself from existing art that relies on the use of an‘optical mask’ (Dulcey et al., Science 252:551 (1991) and U.S. Pat. Nos.5,965,305 and 5,391,463) or ‘contact imprinting’ (U.S. Pat. Nos.5,512,131 and 5,776,748). The use of optical masks for protecting orde-protecting defined regions of a substrate is limited to the use ofphotoactivatible chemistries and/or photolabile molecules. The use of‘contact imprinting’ is limited to solution phase transfer of materialsonto a surface while not enabling ‘protection’ or ‘de-protection’ ofdefined regions of the surface. Further, contact imprinting does notenable reproducible transfer of controlled amounts of material onto thesurface. The use of a ‘stencil’, as disclosed in this invention, allowsfor protection of a region of the substrate to enable modification ofunprotected regions with solution or vapor phase chemistries not limitedto photoreactive/photolabile molecules.

The present invention is not constrained to one particular kind ofsubstrate. The tethering chemistry of the primary monolayer, or theorganosilane, is such that it reacts with surface hydroxyl groups. Thesehydroxyl groups can be introduced on the surface of virtually anyplastic and glass by low temperature plasma treatment. The secondarytethering chemistry, tresyl chemistry, can react with surface amines,hydroxyl, and sulfhydryls, making it possible to attach to a wider arrayof surface chemistry. The desired effect is also achievable with highdensity surface hydroxyl groups, (which may eliminate any silanetreatment). (Dust, Macromolecules, 1990. 23:3742-3746; U.S. Pat. No.5,330,911) All of these benefits make the disclosed method of patterningon glass and plastics affordable, facile, and accurate.

The benign nature of the chemistry employed makes it attractive forbiological applications, allowing the array to be prepared on glass andany thermoplastic and thermoset of choice including, but not limited topoly(styrene), poly(olefin), poly(dimethyl) siloxane (PDMS),poly(carbonate), poly(vinyl) chloride, poly(ethylene), poly(ethylene)terapthalate, Teflon, and fluoronated ethylene co-poly(propylene) (FEP).The present methods also have the ease and flexibility to be applied topolymeric and glass substrates using the same method. Plastics such aspoly(styrene), acrylics, and poly(olefin) have benefits over glass,ceramics and metals because of their affordability, flexibility of shapeand size, ease of engineering, durability, low cost and control over itsoptical quality. The plastics are easily obtained at a minimal cost, canbe molded into almost any shape conceivable, and are durable.

The present methods for preparing a substrate for selective cellpatterning are more reproducible than are methods that employ contactprinting, because there is less opportunity for operator error. There isoperator dependence when contact printing due to the subjectivity ofapplying the stamp to the substrate (force by which the stamp isdepressed, amount of solution on the stamp) and so the results willvary. (U.S. Pat. No. 5,776,748) The present method of using a stencilfor masking while performing solution or vapor phase deposition of thecell adhesive molecules and/or cell repulsive moieties is operatorindependent, thus providing a scalable and manufacturable process.

The instantly disclosed method of cell patterning has a marked advantageover prior thiol chemistry. Previous technology of contact printing withthiols not only introduces operator error, but also requires a thinlayer of gold to be evaporated on the surface of the tissue culturesubstrate. Due to the high temperature involved with gold evaporation,most plastics cannot be used. Optical quality is constrained andfluorescence light is absorbed due to the added layer of gold, whichreduces the quality of information gathered when conducting cell-basedscreening. In addition to a lower optical quality, there is a high costassociated with gold coating. Furthermore, silane linkages are covalent,and are not subject to degradation, as are thiols on gold, which degradeover time due to impurities and the fact that a thiol bond is coordinateand not covalent. The methods of the present invention permit cellpatterning on an optically clear substrate and give the added option ofcontrol over the substrate, so that one has the freedom to choose themost superior affordable plastic or glass for optical quality.

In a particular embodiment of the present method, oxygen plasma is usedto activate the surface in the case of poly(styrene) and poly(olefin),and acid washing is used to activate the surface in the case of glass.Both surfaces can be further incubated with a mildly acidic alcoholicsolution of aminosilane featuring a primary amine on the terminating endof the tethered molecule. Following silane treatment, a stencil isapplied to the substrate. An aqueous solution of tresylchloride-activated PEG is applied to the substrate around the stencilresulting in regions of exposed amine, and regions of PEG in carefullycontrolled proximity to one another. After surface modification, thesurface can be primed with cell adhesive proteins, protein fragments, orpeptides to speed the cell adhesion process. (U.S. Pat. No. 5,874,219)

In another aspect, the present invention provides novel patternedsubstrates for cell culture. In one aspect, the invention provides cellpatterning substrates, comprising:

1. at least a first portion having a reactive surface to which aplurality of cell adhesive molecules are coupled;

2. and at least a second portion having an exposed surface to which aplurality of cell repulsive moieties are coupled; wherein the celladhesive molecules are selected from the group consisting of silanes,and wherein the cell repulsive moieties comprise tresyl-chlorideactivated poly(ethylene) glycol.

In a preferred embodiment, the silane comprises R_(n)SiX_(4-n), wheren=1, 2, or 3; X═Cl, OCH₃, or OC₂H₅; R=a nucleophile, including but notlimited to sulfhydryl groups, amine groups, hydroxyl groups, chargedgroups, polar groups, or proteins, protein fragments, peptides, andsynthetic ligands for cell surface receptors, wherein the nucleophilecan bind to other molecules and/or cells. In a preferred embodiment, thesilane is an aminosilane. In a more preferred embodiment, theaminosilane is selected from the group consisting of methoxy or ethoxysilanes, which include but are not limited totrimethoxysilylpropyldiethylenetriamine, trimethoxysilylethylenediamine,aminopropyltriethoxysilane, trimethoxyaminopropyl-silane, orchlorosilanes such as trichlorosilylethylenediamine,aminopropyltrichlorosilane. In a most preferred embodiment,trimethoxysilylpropyldiethylenetriamine is used.

In another embodiment, the substrate further comprises cell adhesiveproteins, protein fragments, or peptides, including but not limited tofibronectin, laminin, collagen, vitronectin, osteopontin, RGD peptides,RGDS peptides, YIGSR peptides. The strength of cell adhesion to the celladhesion promoters can be modified by varying the composition of thecell adhesive proteins, protein fragments, or peptides. In a furtherembodiment, the substrate further comprises cells bound to the cellbinding locations, either directly or indirectly via cell adhesiveproteins, protein fragments, or peptides. Any cell type may be used,including prokaryotic, eukaryotic, and archaebacterial cells.

The cell binding locations according to the various methods andsubstrates of the invention can be as small as the diameter of a singlecell and as large as several hundred cell diameters. The distancebetween cell binding locations (i.e.: the cell repulsive locations) iscell size dependent, but is sufficiently large so that a cell cannotbridge the gap between cell binding locations (i.e.: 1 cell diameter),unless a particular application calls for interaction of cells indifferent cell binding locations.

In a further embodiment, the various cell patterning substrates aremated with a fluid delivery system to provide fluid and/or reagent flowto the cell binding location. In a preferred embodiment, the fluiddelivery system is that described herein.

In another embodiment, the cell patterning substrate comprises a cellpatterning substrate made by the methods of the invention, as disclosedabove.

This aspect of the present invention may be better understood withreference to the accompanying preferred embodiments that are intendedfor purposes of illustration only and should not be construed to limitthe scope of the invention, as defined by the claims appended hereto.

Materials and Methods:

Reagents and instrumentation that can be utilized in carrying out themethods of the invention include, but are not limited to, 60 and 35 mmpetri dishes, microplates, thermoplastics, poly(olefin), plasmacleaner/sterilizer, digital convection gauges,trimethoxysilylpropyldiethylenetriamine, and2,2,2-trifluoroethanesylphonyl-poly(ethylene)₅₀₀₀ glycol.

Poly(styrene), poly(olefin), or other thermoplastic substrates such aspoly(esters) and poly(ether) are oxygen plasma treated inside a plasmacleaner using the following method. Substrates are placed inside theglass tube chamber and the chamber is evacuated to a pressure of ˜200mtorr as indicated by a convection gauge. Oxygen is pulsed in through aregulation valve and the chamber is evacuated again to a pressure of˜200 mtorr. The above oxygen pulse is repeated 2 more times. After thelast oxygen pulse, the gas is allowed to bleed constantly into thechamber, and the final equilibrium pressure (with the oxygen bleed valveon and the vacuum pump activated) should be ˜300 mtorr. After the properpressure is reached, the voltage switch is turned up to HI (100W) andthe substrates are treated for 25 minutes.

Glass surfaces are activated using the following method. A 1M KOHsolution is prepared in double distilled/deionized (DI) water. The glasssurfaces are incubated for 10 minutes in 1M KOH. After 10 minutes, thesubstrates are rinsed 3 times in double DI water. Coverslips are soakedin HCl:MeOH (1:1) for 30 minutes. After the incubation, the coverslipsare rinsed in double DI water, and transferred into a concentrated bathof sulfuric acid for 30 minutes, followed by 3 rinses with double DIwater. The coverslips are then boiled in distilled water for 15 minutes,and the surfaces blown dry with a nitrogen gun.

Aminosilane treatment is the same for glass, poly(styrene), andpoly(olefin). A 1% solution of trimethoxysilylpropyldiethylenetriamineis prepared in mildly acidified methanol (94% methanol, 5% water, and0.004% glacial acetic acid) and incubated with the substrates for 15minutes. Following silane treatment, the substrates are rinsed withmethanol and baked in a 80° C. oven for 30 minutes.

The PDMS stencil is applied to the aminated glass or poly(styrene) (thisembodiment includes but is not limited to 50, 100, 200 micron and 500micron spots). Pressure is applied until the stencil makes a tight seal.

Tresyl-PEG treatment is the same for glass and poly(styrene). Afterstencil application, a 0.12M sodium bicarbonate solution is prepared inwater. A 25% solution of tresyl-PEG (by weight) is prepared in thebicarbonate. The solution is applied around the stencil and allowed topool around the PDMS, resulting in the liquid touching only exposedaminated surface areas. The substrates are incubated for 4 hours.

Following PEG treatment, the surfaces are rinsed with the 0.12M sodiumbicarbonate solution. The substrates are allowed to incubate for 2 hoursand rinsed under a stream of PBS. The substrates (“partially active”)can be stored for more than 30 days in a dry box before protein coatingand cell incubation.

As used herein, the term “partially active” substrates are glass orplastic substrates chemically and/or texturally modified to yield apatterned array of protein binding locations separated by cytophobicdomains.

Protein Coating

1. The partially active substrates are incubated in a protein, proteinfragment, or peptide solution, including but not limited to fibronectin,laminin, collagen, vitronectin, osteopontin, or fragments thereof, aswell as RGD peptides, RGDS peptides, YIGSR peptides, at concentrationsranging from 1 μg/ml to 25 μg/ml, for 1-2 hours.

2. Post-incubation, the substrates are rinsed in a stream of PBS.

3. The protein/peptide coated substrates are lyophilized to preserve theprotein/peptide structure. These “quasi-active” substrates can be storedin a dry box for >30 days. The silane linkages are covalent and notsubject to degradation, as are thiols on gold, which degrade over timedue to impurities and the fact that a thiol bond is coordinate and notcovalent. As used herein, the term “quasi-active” substrates are glassor plastic substrates chemically and/or texturally modified to yield apatterned array of protein or peptide-rich domains separated bycytophobic domains.

Cell Seeding

1. Cells are prepared for seeding by standard techniques.

2. Partially active or quasi-active substrates are re-hydrated in cellculture medium for 5 minutes.

3. The cells are incubated at any desired seeding density with thepartially or quasi active substrate for 30 minutes to 2 hours (dependingon the cell type) in complete cell culture media at 37° C. and 5% CO₂.

4. The “fully active” substrates, arrayed with the cells, can then beused for cell screening assays. After cryopreservation or roomtemperature desiccation the fully active substrates can be stored longterm. As used herein, the term “fully active” substrates are glass orplastic substrates chemically and/or texturally modified to yield apatterned array of cells separated by cytophobic domains. They can bederived from either partially or quasi-active substrates, although theuse of quasi-active substrates is preferred.

EXAMPLE 5 Patterned Stem Cell and Differentiated Cell Substrates

Stem cells possess the intrinsic ability to: (1) undergo self renewal,or (2) produce differentiated progeny. Extrinsic factors (culturemedium, growth factors, physico-chemical cues from the surroundingcellular milieu) mediate the developmental fate of stem cells. Tissuespecific stem cells, also called determined stem cells, also exhibitpluripotency but not totipotency. Determined stem cells provide theability to access a primary, partially committed cell that can be drivento either self-replication in culture, or selectively differentiatedinto a multitude of tissue specific progeny that in-vitro are close,genotypically and phenotypically, to the whole organism, in contrast toimmortalized cells that are both genotypically and phenotypically farremoved from their precursor cells in the organism.

The pharmacological relevance of using stem cell derived tissue-specificprogeny over immortalized cells results from the genotypic andphenotypic match afforded by the former. As a new chemical compound goesfrom being identified as “active” in a primary drug screen, it must passthrough numerous tests designed to assess its pharmacological profile.Indices of bio-relevance such as cytotoxicity, bioavailability, andspecificity are evaluated together with potency to generate thisprofile, and require the use of cells genotypically and phenotypicallymatched to the organism. This increases the probability that thecompound will be therapeutically relevant when it gets to the clinicbeyond the animal testing stage. The use of primary cells (hepatocytes,neurons, chondrocytes, myocytes, adipocytes) are well documented insecondary and tertiary testing. However, the difficulty in obtaining theproper cells (access or amount), and culturing them in sufficientquantity to cover assay capacity remains a major problem. The use ofstem cell derived primary cells can afford a solution to both higherrelevance and a source of primary cells.

Other work has focused on the production of spatially orientedneo-vascular capillaries from endothelial cells that are bound to celladhesion promoters patterned on a substrate. (Rudolph et al., U.S. Pat.No. 5,721,131) However, the resulting substrate contained only a singletype of differentiated cell, because the method did not permitindividually addressing the cell binding locations with differentiatingreagents.

The present invention provides methods for preparing patterned cellsubstrates comprising a multitude of terminally differentiated cellsfrom an ordered array of stem cells, as well as the substratesthemselves. The use of these patterned cell substrates for drugdiscovery increases the confidence level and relevance of the in-vitropharmacological screening data for extrapolation to in-vivo settings.

The methods and substrate of the present invention mimic events indevelopmental biology: formation of committed mature or terminallydifferentiated cells from stem cells using controlled delivery ofdifferentiation factors (including, but not limited to extracellularmatrix (ECM) derivatized substrates, autocrine/paracrine/endocrinefactors, etc.). Prior technology teaches induction of cellularheterogeneity by “peppering” a cell substrate surface with a multitudeof cell adhesive domains enabling selective adhesion of one or moreimmortalized, genotypically and phenotypically distinct cell types fromsolution. The limited number of “cell-specific cell adhesive moieties”limits the number of distinct cell types attainable on a singlesubstrate. Prior technology does not take advantage of the intrinsicability of stem cells to differentiate into a multitude ofdifferentiated progeny.

In contrast, the present invention teaches induction of cellularheterogeneity on any substrate of choice (ceramics, metals, polymers,composites etc.) by the controlled differentiation of population(s) ofstem cells into a multitude of differentiated cells. The controlleddifferentiation enables creation of “patterns of mixed cell types” inany number of variations and geometry. The present invention alsoenables creation of organ specific and tissue specific cell substratesthat are close in genotypic and phenotypic relevance to the organism ofchoice, as well as substrates that model in vivo interactions betweentissue specific and/or organ specific cells. This enables creation of acell based screening platform capable of providing information withgreater relevance to the organism or systemic level.

The preparation cell substrate of the present invention requiresproviding a patterned stem cell substrate, comprising:

(a) a substrate surface;

(b) a plurality of cell binding locations on the surface, wherein a cellbinding location comprises:

-   -   (1) one or more cell adhesive molecules; and    -   (2) a plurality of stem cells bound to the cell adhesive        molecules; and    -   (c) a plurality of cell repulsive locations on the surface,        wherein the cell repulsive locations comprise a cell repulsive        moiety, wherein individual cell binding locations are separated        by cell repulsive locations.

A single type, or multiple types of stem cells are bound to the cellbinding locations in a patterned array. As used herein, the term“patterned stem cell substrate” means that the stem cells are arrayed onthe substrate in a controlled pattern. The stem cells can comprise asingle class of stem cells, or may comprise multiple stem cell types, inwhich case the positioning of the different stem cell types is alsocontrolled.

As used herein, the term “stem cells” refers to any cell type thatpossesses the ability to produce at least one type of differentiatedprogeny. As such, stem cells include, but are not limited to cellscapable of differentiation into any cell type and self-renewal(pluripotent cells); cells capable of differentiation into any cell typebut not self-renewal (totipotent cells); cells capable of self-renewaland differentiation into many cell types (broad potential; multipotentstem cells), cells capable of limited self-renewal and differentiationinto limited types of cells (progenitor cells); and cells that havecommitted to differentiation into a specific cell type, but have not yetcompleted differentiation (committed progenitor cells). (See, forexample, Gage et al. Science 287:1433-1438 (2000).) In variousembodiments, the stem cells are selected from the group consisting ofneural stem cells, neural progenitor cells, glial progenitor cells,mesenchymal stem cells, hematopoietic stem cells, epithelial stem cells,hepatic stem cells, embryonic stem cells, or combinations thereof.Reference to specific classes of stem cells is provided below:

Hepatic stem cells are reviewed in Thorgeirsson, FASEB J. 10:1249(1996). Examples of hepatic stem cells include, but are not limited to,SEC cells, oval cells, as well as cultured WB-F344 cells, L2039 cells,and RLEΨ13 cells (Coleman et al., Am. J. Pathol. 151:353 (1997); Omoriet al., Hepatology 26:720 (1997); Fiorino et al., In Vitro Cell Dev.Biol. Anim. 34:247-258 (1998); Agelli et al., Histochem. J. 29:205-217(1997); Brill et al., Proc. Soc. Exp. Biol. Med. 204:261-269 (1993)).

Neural stem and progenitor cells: Examples of neuronal stem andprogenitor cells include, but are not limited to, NT-2 cells (Pleasureet al., J. Neuroscience 12:1802-1815 (1992)), and those described inGage, Science 287:1433-1438 (2000); Gritti et al., J. of Neuroscience16:1091-1100 (1996); Frederiksen et al., Neuron 1:439 (1988); Reynoldsand Weiss, Science 255:1707 (1992); Davis and Temple, Nature 372:263(1994); McKay, Science 276:66-71 (1997); Vicario-Abejon et al., Neuron15:105 (1995); Johe et al., Genes and Develop. 10:3129-3142 (1996); andU.S. Pat. Nos. 5,824,489; 6,001,654; 6,033,906).

Glial progenitor cells: Examples of glial progenitor cells include, butare not limited to SNB-19 cells and oligodendrocytes that can bediferentiated in vitro (Raible et al., J. Neurosci. Res. 34:287-294(1993); (Welch et al., In Vitro Cell. Dev. Biol.-Animal 31:610-616(1995)).

Mesenchymal stem cells are pluripotent progenitor cells that possess theability to differentiate into a variety of mesenchymal tissue, includingbone, cartilage, tendon, muscle, marrow stroma, fat and dermis. Suchstem cells include, but are not limited to, C2C12 cells (Teboul et al.,J. Biol. Chem. 270:28183-28187 (1995); Nishimura et al., J. Biol. Chem.273:1872-1879 (1998)); and cells such as those described in U.S. Pat.Nos. 5,486,359; 5,827,740; 5,942,225; 6,022,540.

Hematopoietic stem cells refer to any hematopoietic pluripotentprogenitor cells capable of giving rise to a variety of differentiatedhematopoietic cell types. Cell types within this definition include, butare not limited to CD34⁺ bone marrow mononuclear cells (BMMC) (Berardi,et al., Blood 86:2123-2129, 1995), PBSC (Fritsch, et al., Bone MarrowTransplantation 17:169-178, 1996), cobblestone area forming cells (CAFC)(Lemieux, et al., Blood 86:1339-1347, 1995) and 5-FU BM cells (Alcornand Holyoake, Blood Reviews 10:167-176, 1996); U.S. Pat. No. 5,807,744)

Epithelial stem cells refer to cells that are long-lived, relativelyundifferentiated, have a great potential for cell division, and areultimately responsible for the homeostasis of epithelium. Cells of thistype include, but are not limited to, those described in U.S. Pat. No.5,556,783; U.S. Pat. No. 5,423,778; Rochat et al., Cell 76:1063 (1994);Jones et al. Cell 73:713 (1993); Jones et al., Cell 80:83 (1995)) andSlack, Science 287:1431-1433 (2000).

The cell adhesive molecules of this aspect of the invention can compriseany compound that is capable of supporting stem cell adhesion to thesubstrate, such as those described above, including cell adhesionmolecules, co-polymer blends of extracellular matrix proteins or proteinfragments such as RGD-containing peptides, silanes or thiols. In oneembodiment, aminosilanes, such as methoxy or ethoxy silanes as disclosedabove, are used.

The cell repulsive moieties comprise moieties that are capable ofdirectly inhibiting cell binding, or that bind to other moieties whichinhibit cell binding to the cell repulsive location, including but notlimited to polyethylene glycol (PEG) and other oxygen-rich compounds,sugars, hydrogels, extremely hydrophilic surfaces, or extremelyhydrophobic surfaces, as described above.

The cell binding locations may also comprise inhibitors of uncontrolleddifferentiation of the stem cell, which can comprise any compound knownin the art to prevent uncontrolled stem cell differentiation. Suchcompounds include, but are not limited to self-assembled monolayers ofthiols or silanes coupled to cell adhesive ligands, which are utilizedto enable the creation of cell binding locations, while preventing theiruncontrolled differentiation. Another example is the addition ofprotease thrombin to cultures of the Neuro2A neuroblastoma cell line,which inhibits differentiation of the cells into neurite-containingneuronal cells. (Gurwitz et al., Proc. Natl. Acad. Sci. 85:3440-3444(1988)) Thus, the cell binding promoter can also serve as an inhibitorof uncontrolled differentiation. Alternatively, the cell bindinglocations are covered with a feeder layer of cells to inhibituncontrolled stem cell differentiation.

The substrate of this aspect of the invention can be made of anymaterial known in the art, including but not limited to plastics, glass,ceramics and metals. Preferably, such patterned substrates are made oncommercially viable plastic substrates such as polystyrene orpoly(olefin). In a preferred embodiment, the substrates possess 100μm-500 μm diameter circular cell binding locations separated byedge-to-edge spacing ranging from 25 μm to 125 μm. The overall footprinton the substrate will match the format of 96, 384, and 1536 wellmicroplates, and be approximately 150-fold smaller in surface areacompared to 1536 (32 rows×48 columns), 384 (16 rows×24 columns), and 96wells (8 rows×12 columns) microplates.

In a further aspect, the present invention provides a patterneddifferentiated cell substrate comprising:

(a) a substrate surface;

(b) a first plurality of cell binding locations on the surfacecomprising:

-   -   (i) one or more cell adhesive molecule;    -   (ii) a first differentiated cell type;

(c) a second plurality of cell binding locations on the surfacecomprising:

-   -   (i) one or more cell adhesive molecule;    -   (ii) a second differentiated cell type; wherein the first and        second differentiated cell types are arrayed on the substrate in        a controlled pattern; and

(d) a plurality of cell repulsive locations on the surface, wherein thecell repulsive locations comprise a cell repulsive moiety, whereinindividual cell binding locations are separated by cell repulsivelocations.

As used herein, the term “patterned differentiated cell substrate” meansthat the differentiated cells are arrayed on the substrate in acontrolled fashion, wherein the terminal differentiation is achievedpost-patterning.

In one embodiment, the differentiated cells are derived from stem cellsthat were selectively differentiated on the substrate. In thisembodiment, the stem cell type is selected from the group consisting ofneural stem cells, neural progenitor cells, glial progenitor cells,mesenchymal stem cells, hematopoietic stem cells, epithelial stem cells,hepatic stem cells, embryonic stem cells, or combinations thereof.Alternatively, differentiation can be initiated prior to plating thecells (committed progenitor cells) on the substrate. One of skill in theart will be able to envision many permutations of this embodiment,including but not limited to the following:

1. Selective differentiation of a single stem cell type into two or moredifferentiated cell types in predetermined locations on a singlesubstrate.

2. Selective differentiation of two or more stem cell types arrayed on asingle substrate to generate two or more differentiated cell types.

3. Sequential, selective differentiation of a single or multiple stemcell types, whereby a single differentiated cell type is produced first,followed by selective differentiation of the stem cell type(s) in othercell binding locations.

Thus, in a preferred embodiment, the present invention results in asubstrate with multiple types of differentiated cell types arranged in apre-determined manner. The number of different cell types that can bearrayed is limited only by the differentiation potential of the stemcell, since the various cell binding locations can be individuallyaddressed with differentiating agents, using devices including, but notlimited to microspotters (Cartesian Technologies™, Hewlett Packard™,Genetic MicroSystems™), and fluid delivery system such as, but notlimited to those disclosed herein, and in U.S. Pat. Nos. 5,858,188; and6,007,690. Selective addressing of the stem cells with differentiatingagents enables controlled differentiation into the progeny of choice. Ina preferred embodiment, the fluid delivery system of the presentinvention is combined with the patterned cell substrate to produce amicrofluidic cassette, which can deliver differentiating compounds tothe patterned undifferentiated stem cells.

In a preferred embodiment, the first and second differentiated celltypes are cell types found in a single tissue, including but not limitedto brain, vascular tissue, skin, pancreas, kidney, liver, lung, heart,intestine, and stomach.

In another preferred embodiment, the first and second differentiatedcell types are cell types that are found in different tissues thatinteract in vivo, including but not limited to peripheral nerve-smoothand/or skeletal muscle; epithelial tissue-smooth muscle; vascularendothelium-smooth muscle; glial cells-endothelial cells of bloodcapillaries; adipose cells-axons of peripheral nerve cells and/orSchwann cells; and pancreatic B cells-pancreatic A cells.

In another preferred embodiment, the first and second differentiatedcell types are selected from the group consisting of glial cells,neurons, adipocytes, smooth muscle cells, skeletal muscle cells,osteoblasts, chondrocytes, stromal cells, and myocytes.

In various most preferred embodiments, (a) the first differentiated celltype derived is a glial cell, and the second differentiated cell type isa neuronal cell, thus producing a cell substrate model for the brain;(b) the first differentiated cell type is an adipocyte and the seconddifferentiated cell type is a muscle cell; (c) the first differentiatedcell type a neuronal cell and the second differentiated cell type is amuscle cell.

In a further preferred embodiment, the patterned stem cell substrate orthe patterned cell substrate is mated with a fluid delivery systemchamber to form a cassette, wherein the fluid delivery system deliversreagents to the stem cells or differentiated cells, and comprises:

-   -   (i) a plurality of domains matching the cell binding locations        on the surface of the substrate, and    -   (ii) microfluidic channels that supply reagents to the cell        binding locations.

As used herein, “reagents” include differentiating agents, as well ascell culture medium and any cell culture supplements for cell culture,and test compounds for screening the effects of drug compound librariesand toxins on the cells.

In a most preferred embodiment, a single microfluidic channel suppliesfluid to a single cell binding location on the substrate, to provideseparate fluid flow to each cell binding location, thereby permittingselective differentiation of the stem cells, and/or selective treatmentof the stem cells or differentiated cells in one or more cell bindinglocations with a test agent of choice.

In another aspect, the present invention provides a patterneddifferentiated cell substrate made by a method of selectivedifferentiation of patterned stem cells, wherein the method comprises:

(a) providing a patterned stem cell substrate, comprising

-   -   (1) a substrate surface;    -   (2) a first plurality of cell binding locations on the surface        comprising        -   (i) one or more cell adhesive molecules;        -   (ii) a first differentiated cell type;    -   (3) a second plurality of cell binding locations on the surface        comprising:        -   (i) one or more cell adhesive molecules;        -   (ii) a second differentiated cell type; wherein the first            and second differentiated cell types are arrayed on the            substrate in a controlled pattern;

(b) selectively contacting the cell binding locations withdifferentiating agents to provide controlled differentiation of the stemcells into a progeny of choice, wherein the selective contactingproduces a patterned differentiated cell substrate.

In a preferred embodiment, the patterned cell substrate is mated with afluid delivery system as described above.

-   -   In further preferred embodiments of the above aspects, the stem        cells and the resulting differentiated cells possess at least        one luminescent reporter molecule, for use in cell screening        assays to determine the distribution, environment or activity of        the luminescent reporter molecules on or in the cells or within        or between subcellular compartments of the cells, in response to        a test agent of choice. A variety of such luminescent reporter        molecules can be used in this aspect of the invention, including        but not limited to those described in U.S. Pat. No. 5,989,835;        pending U.S. patent application Ser. Nos. 09/031,271 (filed Feb.        27, 1998); 09/352,171 (filed Jul. 12, 1999); 09/293,209 (filed        Apr. 16, 1999); 09/293,209 (filed Apr. 16, 1999); 09/398,965        (filed Sep. 17, 1999); 09/430,656 (filed Oct. 29, 1999);        09/513,783 (filed Feb. 24, 2000); Giuliano et al., Ann. Rev.        Biophys. Biomol. Struct. 24:405-434 (1995); and Giuliano et al.,        Trends in Biotech. 16:135-140 (1998), all references        incorporated by reference herein in their entirety.    -   The luminescent probes can be small molecules, labeled        macromolecules, or genetically engineered proteins, including,        but not limited to green fluorescent protein chimeras. As used        herein, “luminescent” probes include fluorescent, luminescent,        and chemiluminescent probes.    -   In another embodiment, only one of the differentiated cell types        possesses a luminescent reporter molecule, and when an        interaction occurs between the first and second differentiated        cell types, only one of the cell types reports the interaction,        via the luminescent reporter molecule.

The luminescently labeled reporter molecule may be expressed by or addedto the cells either before, together with, or after contacting the cellswith a test compound. For example, the reporter molecule may beexpressed as a luminescently labeled protein chimera by transfected stemcells. Alternatively, the luminescently labeled reporter molecule may beexpressed, isolated, and bulk-loaded into the stem cells, or thereporter molecule may be luminescently labeled after isolation. As afurther alternative, the reporter molecule can be expressed by the stemcell, which is subsequently contacted with a luminescent label, such asa labeled antibody, that detects the reporter molecule.

Preferably, the luminescent reporter molecules in the firstdifferentiated cell type are spectrally distinguishable from theluminescent reporter molecules in the second differentiated cell type.

The present invention also provides methods for selective stem celldifferentiation, comprising providing a patterned stem cell substrate asdisclosed above, and selectively contacting the cell binding locationswith differentiating agents to provide controlled differentiation of thestem cells into the progeny of choice, wherein the selective contactingproduces a patterned differentiated cell substrate. Any method known inthe art for differentiating the stem cells into differentiated cells canbe used. References providing conditions for differentiating the variousstem cells into differentiated progeny can be found, for example, in thefollowing references, which are incorporated by reference herein intheir entirety:

Mesenchymal stem cells: U.S. Pat. No. 5,827,740 (adipogenicdifferentiation); U.S. Pat. No. 6,022,540 (osteogenic differentiation);U.S. Pat. No. 5,942,225 (osteogenic, chondrogenic, tendenogenic, marrowstromal cell, and myogenic differentiation); Cuenda et al., J. Biol.Chem. 274:4341-4346 (1999) (C2C12 myogenic differentiation); Nishimuraet al., J. Biol. Chem. 273:1872-1879 (1998) (C2C12 osteoblasticdifferentiation); Teboul et al., J. Biol. Chem. 270:28183-28187 (1995)(C2C12 adipogenic differentiation)

Neural stem and progenitor cells: U.S. Pat. No. 5,824,489 (neurons andglia); U.S. Pat. No. 6,001,654 (neurons and smooth muscle cells); U.S.Pat. No. 6,033,906 (glial cells); Pleasure et al., J. Neurosci.12:1802-1815 (1992) (NTera2 differentiation into neurons);

Glial and neural stem and progenitor cells: Welch et al., In Vitro Cell.Dev. Biol.-Animal 31:610-616 (1995) (SNB-19 glial differentiation); U.S.Pat. No. 5,824,489 (neurons and glia).

Hematopoietic stem cells: Lawman et al., J. Hematother. 1:251-259(1992); Huss, Stem Cells 18:1-9 (2000); Zhang et al., Blood 95:138-146(2000); Zhang et al., Blood 92:118-128 (1998).

Hepatic stem cells: Brill et al., Proc. Soc. Exp. Biol. Med. 204:261-269(1993); Brill et al., Dig. Dis. Sci., 44:364-371 (1999); Fiorino et al.,In Vitro Cell Dev. Biol. Anim. 34:247-258 (1998).

In various preferred embodiments, the substrate is mated with a fluiddelivery system as disclosed above, and the stem cells possess at leastone luminescent reporter molecule that serves to identify the phenotypeof the progeny of choice.

In these embodiments, the stem cells are contacted with differentiatingagents, to effect differentiation of the transfected patterned cellsinto differentiated progeny, using the appropriate differentiatingagents applied either homogeneously to the substrate (for singleprogeny) or selectively to specific cell binding locations (multipleprogeny). Differentiation of a single stem cell type into differentprogeny, or of different stem cells into different progeny, permits theformation of substrates with cell binding locations that bear differentcell types in any desired juxtaposition. In this way, simple tomoderately complex models of cellular differentiation are used toprepare multicellular tissue-specific and organ-specific cell substratesfor use in cell based analysis for drug discovery and life sciences.

In one model system, neuronal and glial stem cells are engineered toexpress fluorescent protein reporter molecules to measure the dynamicsof their cytoskeletal proteins. The cytoskeleton has become awell-characterized and valid drug discovery target for which there arelikely to be many lead compounds in the drug discovery pipeline at anyone time. Each of the stem cell lines express spectrally distinctreporter molecules such that they can be patterned into separatelocations within a cell array as well as be patterned together,(co-cultured) within the same location. Alternatively, the differentstem cells may be patterned into separate locations, wherein thelocations are in close enough proximity to permit interactions betweenthe cells in the different locations. The latter two aspect allow thesimultaneous measurement of drug responses of the two cell types in anorganotypic context where the cells are allowed to interact as theywould within the brain tissue of a living animal. Because the reportermolecules contained within each cell type are spectrally distinct, theplatform detects and assigns function to each cell type within theco-culture.

In a second model system, a pluripotent cell line, mouse C2C12 cells, isengineered with a luminescent reporter molecule, patterned intomicroarrays, and differentiated into two cell types, skeletal musclemyocytes and adipocytes. In this case, the stem cells are engineered toexpress a luminescent reporter molecule of carbohydrate metabolic flux,including but not limited to reporters of the phosphorylation state ofPFK-1 and PFK-2, the measurement of cellular ATP levels (energy charge),the ratio of oxidation-reduction co-factors such as NAD^(+/)NADH, andthe concentration of the second messenger cAMP, that are measured bothin time and in space within each cell type. For muscle and adiposecells, carbohydrate metabolism plays an important role in regulating thephysiological function of each cell type; contraction and relaxation ofthe muscle cells and fat storage and mobilization in adipocytes.Therefore, this model system permits measurement of the effect of leadcompounds on the same molecular pathway within two tissue types.Moreover, this multiple tissue type screening platform permits theefficient addressing of lead compound efficacy, specificity, andtoxicology.

A third, more complex model system involves the co-cultivation ofneuronal and skeletal muscle stem cells with both types being engineeredto express spectrally distinct luminescent reporter molecules. The cellsare patterned and differentiated on the substrate. The effect of leadcompounds on the complex interaction of neurons and skeletal musclecells is measured using the luminescent reporter molecules engineeredinto each cell type. The co-cultivation of differentiated neuronal andmuscle cells permits direct measurement of excitation-contractioncoupling events and the effects that lead compounds have on theseevents.

These approaches can be generalized to interactions between other tissuetypes and interactions between multiple cell types within an organ,including but not limited to peripheral nerve-smooth or skeletal muscle;epithelial tissue-smooth muscle; vascular endothelium-smooth muscle;glial cells-endothelial cells of blood capillaries; adipose cells-axonsof peripheral nerve cells and Schwann cells; and pancreatic Bcells-pancreatic A cells.

The present invention further provides methods for cell based screening,wherein the stem cells and/or differentiated cells possess at least oneluminescent reporter molecule, for use in cell screening assays todetermine the distribution, environment or activity of the luminescentreporter molecules on or in the cells or within or between subcellularcompartments of the cells, in response to a test agent of choice. Avariety of such luminescent reporter molecules can be used in thisaspect of the invention, including but not limited to those described inU.S. Pat. No. 5,989,835; pending U.S. patent application Ser. Nos.09/031,271 (filed Feb. 27, 1998); 09/352,171 (filed Jul. 12, 1999);09/293,209 (filed Apr. 16, 1999); 09/293,209 (filed Apr. 16, 1999);09/398,965 (filed Sep. 17, 1999); 09/430,656 (filed Oct. 29, 1999);09/513,783 (filed Feb. 24, 2000); Giuliano et al., Ann. Rev. Biophys.Biomol. Struct. 24:405-434 (1995); and Giuliano et al., Trends inBiotech. 16:135-140 (1998), all references incorporated by referenceherein in their entirety.

In this embodiment of the method, the patterned stem cell arrays andpatterned differentiated cell arrays are used to analyze changes in thedistribution, environment or activity of the luminescent reportermolecules on or in the cells or within or between subcellularcompartments of the cells in response to a test compound. The cells areimaged and/or scanned using a cell screening system comprising anoptical system with a stage adapted for holding a substrate containingcells, a detection device that is capable of creating a digital image, ameans for directing fluorescence or luminescence emitted from the cellsto the detection device, and a computer for receiving and processingdata from the detection device. A preferred embodiment of such a deviceis disclosed in U.S. Pat. No. 5,989,835; and pending U.S. patentapplication Ser. No. 09/031,271 (filed Feb. 27, 1998), both referencesincorporated by reference herein in their entirety. Utilizing the cellscreening system, luminescent signals from the reporter molecules areconverted into digital data; and the digital data is used to determinechanges in the distribution, environment or activity of the reportermolecules in response to the test agent.

-   -   Such digital data can be used to report the effect of a test        compound on distribution of the reporter molecule between:        cytoplasm-nucleus, cell membrane-cytoplasm, endoplasmic        reticulum-Golgi apparatus, as well as to report on apoptosis;        receptor internalization; transcription factor activation;        protein kinase activation; protease activity; organelle        structure, distribution, and function; macromolecule        distribution; gene expression; microtubule cytoskeletal        structure; actin cytoskeletal structure; nuclear shape; nuclear        area; nuclear size; nuclear perimeter; mitochondrial potential;        cell shape; cell motility; cell size; and cell perimeter. (For        example, see U.S. Pat. No. 5,989,835; pending U.S. patent        application Ser. Nos. 09/031,271 (filed Feb. 27, 1998);        09/352,171 (filed Jul. 12, 1999); 09/293,209 (filed Apr. 16,        1999); 09/398,965 (filed Sep. 17, 1999); 09/430,656 (filed Oct.        29, 1999); and 09/513,783 (filed Feb. 24, 2000).

The use of a fluid delivery system in the method, including but notlimited to that disclosed above, or the use of automated precisioninstruments such as microspotters (Cartesian Technologies™, HewlettPackard™, Genetic MicroSystems™), permits the delivery of specific cellbinding locations with a differentiating agent of choice.

EXAMPLES

Organotypic Differentiation Model Systems

Glial differentiation: Cells taken from a highly aggressive humanglioblastoma tumor have been shown to grow indefinitely in culture andto exhibit altered morphological and growth characteristics in thepresence of a differentiation agent. These cells, named SNB-19 (Welch etal., 1995), are engineered to express a luminescent reporter molecule(see below) and patterned onto cell substrates, either by themselves orin combination with neuronal stem cells. To induce differentiation, amixture of 1 mM dibutyryl-cAMP and 1 mM isobutylmethyl xanthine (aphosphodiesterase inhibitor) is added to the culture, and the cells areallowed to incubate for 12-24 hours. These agents induce the cells toelaborate multiple processes that often interact with other glia in thesame culture (Welch et al., 1995), as well as cause the cells to stopdividing.

The glial stem cells are transfected to express a green fluorescentprotein (GFP)-glial fibrillary acidic protein (GFAP). chimera. GFAP is acomponent of the intermediate filament cytoskeleton, and is a majorcytoskeletal protein found in glial stem cells and differentiated glia.

Neuronal differentiation: Several neuronal stem cell lines exist thatcan be used in the instant invention. NT2 cells from a humanteratocarcinoma cell line (Pleasure et al., 1992) are unique in thatthey can be induced to differentiate into stable, post-mitotic humanneurons, and they have been shown to be a vehicle for the expression ofdiverse gene products. (Pleasure et al., 1992) NT2 cells are engineeredto express a blue fluorescent protein (BFP)-β-tubulin chimera. β-tubulinis a major component of the cytoskeleton. The cells are then patternedonto cell substrates, either by themselves or in combination with glialstem cells. To induce differentiation, NT2 cells initially enter aprogram of differentiation that begins with a two week treatment ofretinoic acid (10⁻⁵ M), mitotic inhibitors, and a specializedextracellular matrix. The partially differentiated cells are transferredto the substrates where they undergo the final stages of differentiationby elaborating processes that form axons and dendrites. The cells becomepost-mitotic, but retain the ability to express functional proteins,such as the luminescent protein reporter molecule.

Mixed glial-neuronal differentiation: NT2 stem cells in the final stagesof differentiation are added with SNB-19 cells to the same substrate.After both cell types attach, the co-cultures are treated with 1 mMdibutyryl-cAMP and 1 mM isobutylmethyl xanthine. The two cell types areallowed to interact as they differentiate. Because the NT2 cells arecommitted to differentiation, the dibutyryl-cAMP has little to no effecton neuronal cell differentiation, and may even enhance it, since cAMP isknown to induce the differentiation of several cell types.

Adipose and skeletal muscle tissue from a common stem cell: The mouseC2C12 cell line is pluripotent and has been shown capable ofdifferentiating into skeletal muscle (Cuenda and Cohen, 1999),adipocytes (Teboul et al., 1995), and osteoblasts (Nishimura et al.,1998). The C2C12 cells are first engineered to express a luminescentreporter molecule of carbohydrate metabolism (see below). The cells arepatterned onto substrates where the growth medium contains <1% calfserum. This large decrease in serum concentration (10% originally)induces the C2C12 cells, over a period of 24-48 hours, to stop dividing,fuse into elongated, multinucleated cells, and form contractilemyotubes. To induce adipocyte differentiation, the cells are treatedwith a mix of 5 μM thiazolidinedione and 100 μM fatty acid (Teboul etal., 1995). Differentiation occurs after 24-48 hours and is accompaniedby the slowing of cell growth and the uptake of fatty acids by the cellsand their incorporation into lipid droplets.

The C2C12 cells are transfected with a reporter molecule comprising6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2), whichplays a key role in balancing cellular energy utilization and storage.The activity of this bifunctional enzyme can act to switch a cellbetween carbohydrate oxidation (energy yielding) and carbohydratesynthesis (energy requiring). The phosphorylation state of PFK-2dictates whether the enzyme will stimulate cellular carbohydratebreakdown or synthesis. (Kurland and Pilkis, Protein Science 4:1023-1037(1995). The amino acid sequence containing the PFK-2 phosphorylationsite is inserted into a specific site within the coding sequence for GFPthat tolerates insertions (Baird et al., Proc. Natl. Acad. Sci.,96:11241-11246 (1999)), wherein the fluorescence properties of GFP arealtered upon phosphorylation of PFK-2.

Nerve and muscle tissue interaction: Cellular components from the othermodel systems are combined to model tissue-tissue interactions. NeuronalNT2 cells are combined with C2C12 cells to allow their interactionduring differentiation, much like tissues interact develop during normaldevelopment. Three scenarios are tested:

1. NT2 and C2C12 cells are arrayed together and allowed to differentiatetogether;

2. NT2 cells are arrayed on the substrate and differentiated, followedby addition and differentiation of C2C12 cells;

3. C2C12 muscle stem cells are arrayed on the substrate anddifferentiated, followed by addition and differentiation of neuronal NT2stems.

OTHER EXAMPLES

Neural crest stem cells can be treated with poly-D-lysine andfibronectin to produce peripheral neurons and glia, as disclosed in U.S.Pat. No. 6,033,906. The same stem cells when treated with fibronectinonly produce glial cells but not neurons.

Mesenchymal stem cells treated with 100 nM dexamethasone +10 mMβ-glycerophosphate are induced to undergo osteogenesis. (U.S. Pat. No.5,942,225) The same stem cells treated with 5 ng/ml BMP are induced toundergo chondrogenesis; if treated with 5-azacytidine, they are inducedto undergo myogenesis. Finally, the same stem cells treated with 10 u/mlIL-1α differentiate into stromal cells.

U.S. Pat. No. 5,827,740 teaches treating mesenchymal stem cells withglucocorticoids and phosphodiesterase inhibitor to induce adipogenesis.

EXAMPLE 6 Fluorescent Biosensor Toxin Characterization

As used herein, “toxin” refers to any organism, macromolecule, ororganic or inorganic molecule or ion that alters normal physiologicalprocesses found within a cell, or any organism, macromolecule, ororganic or inorganic molecule or ion that alters the physiologicalresponse to modulators of known physiological processes. Thus, a toxincan mimic a normal cell stimulus, or can alter a response to a normalcell stimulus.

Living cells are the targets of toxic agents that can compriseorganisms, macromolecules, or organic or inorganic molecules. Acell-based approach to toxin detection, classification, andidentification would exploit the sensitive and specific moleculardetection and amplification system developed by cells to sense minutechanges in their external milieu. By combining the evolved sensingcapability of cells with the luminescent reporter molecules and assaysdescribed herein, intracellular molecular and chemical events caused bytoxic agents can be converted into detectable spatial and temporalluminescent signals.

When a toxin interacts with a cell, whether it is at the cell surface orwithin a specific intracellular compartment, the toxin invariablyundermines one or more components of the molecular pathways activewithin the cell. Because the cell is comprised of complex networks ofinterconnected molecular pathways, the effects of a toxin will likely betransmitted throughout many cellular pathways. Therefore, our strategyis to use molecular markers within key pathways likely to be affected bytoxins, including but not limited to cell stress pathways, metabolicpathways, signaling pathways, and growth and division pathways.

We have developed and characterized three classes of cell basedluminescent reporter molecules to serve as reporters of toxic threatagents. These 3 classes are as follows:

(1) Detectors: general cell stress detection of a toxin;

(2) Classifiers: perturbation of key molecular pathway(s) for detectionand classification of a toxin; and

(3) Identifiers: activity mediated detection and identification of atoxin or a group of toxins.

Thus, in another aspect of the present invention, living cells are usedas biosensors to interrogate the environment for the presence of toxicagents. In one embodiment of this aspect, an automated method for cellbased toxin characterization is disclosed that comprises providing anarray of locations containing cells to be treated with a test substance,wherein the cells possess at least a first luminescent reporter moleculecomprising a detector and a second luminescent reporter moleculeselected from the group consisting of a classifier or an identifier;contacting the cells with the test substance either before or afterpossession of the first and second luminescent reporter molecules by thecells; imaging or scanning multiple cells in each of the locationscontaining multiple cells to obtain luminescent signals from thedetector; converting the luminescent signals from the detector intodigital data to automatically measure changes in the localization,distribution, or activity of the detector on or in the cell, whichindicates the presence of a toxin in the test substance; selectivelyimaging or scanning the locations containing cells that were contactedwith test sample indicated to have a toxin in it to obtain luminescentsignals from the second reporter molecule; converting the luminescentsignals from the second luminescent reporter molecule into digital datato automatically measure changes in the localization, distribution, oractivity of the classifier or identifier on or in the cell, wherein achange in the localization, distribution, structure or activity of theclassifier identifies a cell pathway that is perturbed by the toxinpresent in the test substance, or wherein a change in the localization,distribution, structure or activity of the identifier identifies thespecific toxin that is present in the test substance. In a preferredembodiment, the cells possess at least a detector, a classifier, and anidentifier. In a further preferred embodiment, the digital data derivedfrom the classifier is used to determine which identifier(s) to employfor identifying the specific toxin or group of toxins.

As used herein, the phrase “the cells possess one or more luminescentreporter molecules” means that the luminescent reporter molecule may beexpressed as a luminescent reporter molecule by the cells, added to thecells as a luminescent reporter molecule, or luminescently labeled bycontacting the cell with a luminescently labeled molecule that binds tothe reporter molecule, such as a dye or antibody, that binds to thereporter molecule. The luminescent reporter molecule can be expressed oradded to the cell either before or after treatment with the testsubstance.

The luminescent reporters comprising detectors, classifiers, andidentifiers may also be distributed separately into single or multiplecell types. For example, one cell type may contain a toxin detector,which, when activated by toxic activity, implies to the user that thesame toxin sample should be screened with reporters of the classifier oridentifier type in yet another population of cells identical to ordifferent from the cells containing the toxin detector.

The detector, classifier, and identifier can comprise the same reportermolecule, or they can comprise different reporters.

Screening for changes in the localization, distribution, structure oractivity of the detectors, classifiers, and/or identifiers can becarried out in either a high throughput or a high content mode. Ingeneral, a high-content assay can be converted to a high-throughputassay if the spatial information rendered by the high-content assay canbe recoded in such a way as to no longer require optical spatialresolution on the cellular or subcellular levels. For example, ahigh-content assay for microtubule reorganization can be carried out byoptically resolving luminescently labeled cellular microtubules andmeasuring their morphology (e.g., bundled vs. non-bundled or normal). Ahigh-throughput version of a microtubule reorganization assay wouldinvolve only a measurement of total microtubule polymer mass aftercellular extraction with a detergent. That is, destabilizedmicrotubules, being more easily extracted, would result in a lower totalmicrotubule mass luminescence signal than unperturbed or drug-stabilizedluminescently labeled microtubules in another treated cell population.The luminescent signal emanating from a domain containing one or morecells will therefore be proportional to the total microtubule massremaining in the cells after toxin treatment and detergent extraction.

The methods for detecting, classifying, and identifying toxins canutilize the same screening methods described throughout the instantapplication, including but not limited to detecting changes in cytoplasmto nucleus translocation, nucleus or nucleolus to cytoplasmtranslocation, receptor internalization, mitochondrial membranepotential, signal intensity, the spectral response of the reportermolecule, phosphorylation, intracellular free ion concentration, cellsize, cell shape, cytoskeleton organization, metabolic processes, cellmotility, cell substrate attachment, cell cycle events, and organellarstructure and function.

In all of these embodiments, the methods can be operated in bothtoxin-mimetic and toxin-inhibitory modes.

Such techniques to assess the presence of toxins are useful for methodsincluding, but not limited to, monitoring the presence of environmentaltoxins in test samples and for toxins utilized in chemical andbiological weapons; and for detecting the presence and characteristicsof toxins during environmental remediation, drug discovery, clinicalapplications, and during the normal development and manufacturingprocess by virtually any type of industry, including but not limited toagriculture, food processing, automobile, electronic, textile, medicaldevice, and petroleum industries.

We have developed and characterized examples of luminescent cell-basedreporters, distributed across the 3 sensor classes. The methodsdisclosed herein can be utilized in conjunction with computer databases,and data management, mining, retrieval, and display methods to extractmeaningful patterns from the enormous data set generated by eachindividual reporter or a combinatorial of reporters in response to toxicagents. Such databases and bioinformatics methods include, but are notlimited to, those disclosed in U.S. patent application Ser. No.09/437,976, filed Nov. 10, 1999; 60/145,770 filed Jul. 27, 1999 and U.S.patent application Ser. No. to be assigned, filed Feb. 19, 2000.(98,068-C)

Any cell type can be used to carry out this aspect of the invention,including prokaryotes such as bacteria and archaebacteria, andeukaryotes, such as single celled fungi (for example, yeast), molds (forexample, Dictyostelium), and protozoa (for example, Euglena). Highereukaryotes, including, but not limited to, avian, amphibian, insect, andmammalian cells can also be used. TABLE 3 Examples of BiosensorsResponse to model toxins # Name Class Cell Types Positive Negative 1Mitochondrial D LLCPK (pig epithelia) Valinomycin Oligomycin PotentialRat primary hepatocytes (10 nM-100 μM) (10 nM) [Donnan Equilibrium FCCPDye] (10 nM-100 μM) 2 Heat Shock D HeLa Cadmium TNF-α Protein 3T3 (10mM) (100 ng/ml) (Hsp 27, Hsp 70) GFP-chimera 3 Tubulin- C BHK PaclitaxelStaurosporine cytoskeleton HeLa (10 nM-20 μM) (1 nM-1 μM) [β-tubulin-GFPLLCPK Curacin-A chimera] (5 nM-10 μM) Nocadazole (7 nM-12 μM) Colchicine(5 nM-10 μM) Vinblastine (5 nM-10 μM) 4 pp38 MAPK-stress C 3T3Anisomycin TNF-α signaling LLCPK (100 μM) (100 ng/ml) [antibody and GFP-Cadmium chimera] (10 μM) 5 NF-κB-stress C HeLa TNF-α Anisomycinsignaling 3T3 (100 ng/ml-0.38 pg/ml) (10 nM-10 μM) [antibody and GFP-BHK IL-1 Cadmium chimera] SNB19 (4 ng/ml-.095 pg/ml) (1-10 μM) HepG2Nisin Penitrem A LLCPK (2-1000 μg/ml) (10 μM) Streptolysin Valinomycin(10 μg/ml) (1 μM) Anisomycin (100 μM) 6 IκB C In many cell types[complement to NF- κB] 7 Tetanus Toxin I In many cell types [Proteaseactivity- based sensor] 8 Anthrax LF I In many cell types [Proteaseactivity- based sensor]Sensor Class:D = Detector of toxins;C = Classifier of toxins;I = Identifier of toxin or group of toxinsThe model toxins can generally be purchased from Sigma Chemical Company(St. Louis, MO)

Examples of Detectors: This class of sensors provides a first linesignal that indicates the presence of a toxic agent. This class ofsensors provides detection of general cellular stress that requiresresolution limited only to the domain over which the measurement isbeing made, and they are amenable to high content screens as well. Thus,either high throughput or high content screening modes may be used,including but not limited to translocation of heat shock factors fromthe cytoplasm to the nucleus, and changes in mitochondrial membranepotential, intracellular free ion concentration detection (for example,Ca²⁺; H⁺), general metabolic status, cell cycle timing events, andorganellar structure and function.

1. Mitochondrial Potential

-   -   A key to maintenance of cellular homeostasis is a constant ATP        energy charge. The cycling of ATP and its metabolites ADP, AMP,        inorganic phosphate, and solution-phase protons is continuously        adjusted to meet the catabolic and anabolic needs of the cell.        Mitochondria are primarily responsible for maintaining a        constant energy charge throughout the entire cell. To produce        ATP from its constituents, mitochondria must maintain a constant        membrane potential within the organelle itself. Therefore,        measurement of this electrical potential with specific        luminescent probes provides a sensitive and rapid readout of        cellular stress.    -   We have utilized a positively charged cyanine dye, JC-1        (Molecular Probes, Eugene, Oreg.), which diffuses into the cell        and readily partitions into the mitochondrial membrane, for        measurement of mitochondrial potential. The photophysics of JC-1        are such that when the probe partitions into the mitochondrial        membrane and it experiences an electrical potential >140 mV, the        probe aggregates and its spectral response is shifted to the        red. At membrane potential values <140 mV, JC-1 is primarily        monomeric and its spectral response is shifted toward the blue.        Therefore, the ratio of two emission wavelengths (645 nm and 530        nm) of JC-1 partitioned into mitochondria provides a sensitive        and continuous measure of mitochondrial membrane potential.    -   We have been making live cell measurements in a high throughput        mode as the basis of a generalized indicator of toxic stress.        The goal of our initial experiments was to determine the ratio        of J-aggregates of JC-1 dye to its monomeric form both before        and after toxic stress.        Procedure

-   1. Cells were plated and cultured up to overnight.

-   2. Cells were stained with JC-1 (10 μg/ml) for 30 minutes at 37° C.    in a CO₂ incubator.

-   3. Cells were then washed quickly with HBSS at 37° C. (2 times, 150    μl/well), the toxins were added if required, and the entire plate    was scanned in a plate reader. The JC-1 monomer was measured    optimally with a 485 nm excitation/530 nm emission wavelength filter    set, and the aggregates were best measured with a 590 nm    excitation/645 nm emission wavelength set.    Results

The mitochondrial potential within several types of living cells, andthe effects of toxins on the potential were measured using thefluorescence ratio Em 645 (590)/Em 530 (485) (excitation wavelengths inparentheses). For example, we measured the effect of 10 μM valinomycinon the mitochondrial potential within LLCPK cells (pig epithelia).Within seconds of treatment, the toxin induced a more rapid and highermagnitude decrease (an approximately 50% reduction) in mitochondrialpotential than that found in untreated cells. Hepatocytes were alsodetermined to be sensitive to valinomycin, and the changes inmitochondrial potential were nearly complete within seconds to minutesafter addition of various concentrations of the toxin.

-   -   These results are consistent with mitochondrial potential being        a model intracellular detector of cell stress. Because these        measurements require no spatial resolution within individual        cells, mitochondrial potential measurements can be made rapidly        on an entire cell array (e.g. high throughput). This means, for        example, that complex arrays of many cell types can be probed        simultaneously and continuously as a generalized toxic response.        Such an indicator can provide a first line signal to indicate        that a general toxic stress is present in a sample. Further        assays can then be conducted to more specifically identify the        toxin using cells classifier or identifier type reporter        molecules.        2. Heat Shock Proteins    -   Most mammalian cells will respond to a variety of environmental        stimuli with the induction of a family of proteins called stress        proteins. Anoxia, amino acid analogues, sulfhydryl-reacting        reagents, transition metal ions, decouplers of oxidative        phosphorylation, viral infections, ethanol, antibiotics,        ionophores, non-steroidal antiinflammatory drugs, thermal stress        and metal chelators are all inducers of cell stress protein        synthesis, function, or both. Upon induction, cell stress        proteins play a role in folding and unfolding proteins,        stabilizing proteins in abnormal configurations, and repairing        DNA damage.    -   There is evidence that at least four heat shock proteins        translocate from the cytoplasm to the nucleus upon stress        activation of the cell. These proteins include the heat shock        proteins HSP27 and HSP70, the heat shock cognate HSC70, and the        heat shock transcription factor HSF1. Therefore, measurement of        cytoplasm to nuclear translocation of these proteins (and other        stress proteins that translocate from the cytoplasm to the        nucleus upon a cell stress) will provide a rapid readout of        cellular stress.    -   We have tested the response of an HSP27-GFP biosensor in two        cell lines (BHK and HeLa) using a library of heavy metal        chemical compounds as biological toxin stimulants to stress the        cells. Briefly, cells expressing the HSP27-GFP biosensor are        plated into 96-well microplates, and allowed to attach. The        cells are then treated with a panel of cell stress-inducing        compounds. Exclusively cytoplasmic localization of the fusion        protein was found in unstimulated cells.    -   Other similar heat shock protein biosensors (HSP-70, HSC70, and        HSF1 fused to GFP) can also be used as detectors.

Examples of Classifiers:

This class of sensors detects the presence of, and further classifiestoxins by identifying the cellular pathway(s) perturbed by the toxin. Assuch, this suite of sensors can detect and/or classify toxins into broadcategories, including but not limited to “toxins affecting signaltransduction,” “toxins affecting the cytoskeleton,” and “toxinsaffecting protein synthesis”. Either high throughput or high contentscreening modes may be used. Classifiers can comprise compoundsincluding but not limited to tubulin, microtubule-associated proteins,actin, actin-binding proteins including but not limited to vinculin,α-actinin, actin depolymerizing factor/cofilin, profilin, and myosin;NF-κB, IκB, GTP-binding proteins including but not limited to rac, rho,and cdc42, and stress-activated protein kinases including but notlimited to p38 mitogen-activated protein kinase.

1. Tubulin-Cytoskeleton

-   -   The cell cytoskeleton plays a major role in cellular functions        and processes, such as endo- and exocytosis, vesicle transport,        and mitosis. Cytoskeleton-affecting toxins, of proteinaceous and        non-proteinaceous form, such as C2 toxin, and several classes of        enterotoxins, act either directly on the cytoskeleton, or        indirectly via regulatory components controlling the        organization of the cytoskeleton. Therefore, measurement of        structural changes in the cytoskeleton can provide        classification of the toxin into a class of        cytoskeleton-affecting toxins. This assay can be conducted in a        high content mode, as described previously, or in a high        throughput mode. For high throughput as discussed previously.    -   Such measurements will be valuable for identification of toxins        including, but not limited to anti-microtubule agents, agents        that generally affect cell cycle progression and cell        proliferation, intracellular signal transduction, and metabolic        processes.    -   For microtubule disruption assays, LLCPK cells stably        transfected with a tubulin-GFP biosensor plasmid were plated on        96 well cell culture dishes at 50-60% confluence and cultured        overnight at 37° C., 5% CO₂. A series of concentrations (10-500        nM) of 5 compounds (paclitaxel, curacin A, nocodazole,        vinblastine, and colchicine) in normal culture media were        freshly prepared from stock, and were added to cell culture        dishes to replace the old culture media. The cells were then        observed with the cell screening system described above, at a 12        hour time point.

Our data indicate that the tubulin chimera localizes to and assemblesinto microtubules throughout the cell. The microtubule arrays in cellsexpressing the chimera respond as follows to a variety ofanti-microtubule compounds: Drug Response Vinblastine DestabilizationNocodazole Destabilization Paclitaxel Stabilization ColchicineDestabilization Curacin A Destabilization

-   -   Similar data were obtained using cells expressing the tubulin        biosensor that were patterned onto cell arrays (such as those        described in U.S. patent application Ser. No. 08/865,341 filed        May 29, 1997, incorporated by reference herein in its entirety)        and dosed as above.        2. NF-κB

NF-κB is cytoplasmic at basal levels of stimulation, but upon insulttranslocates to the nucleus where it binds specific DNA responseelements and activates transcription of a number of genes. Translocationoccurs when IkB is degraded by the proteosome in response to specificphosphorylation and ubiquitination events. IkB normally retains NF-κB inthe cytoplasm via direct interaction with the protein, and masking ofthe NLS sequence of NF-κB. Therefore, although not the initial ordefining event of the whole signal cascade, NF-κB translocation to thenucleus can serve as an indicator of cell stress.

NF-κB Immunolocalization

For further studies, we characterized endogenous NF-κB activation byimmunolocalization in toxin treated cells. The NF-κB antibodies used inthis study were purchased from Santa Cruz Biotechnology, Inc. (SantaCruz, Calif.), and secondary antibodies are from Molecular Probes(Eugene, Oreg.).

For the 3T3 and SNB19 cell types, we determined the effectiveconcentrations that yield response levels of 50% of the maximum (EC50),expressed in units of mass per volume (ng/ml) and units of molarity.Based on molecular weights of 17 kD for both TNFα and IL-1α, the EC50levels for these two compounds with 3T3 and SNB19 cell types are givenin units of molarity in Table 3. Our results demonstratedreproducibility of the relative responses from zero to maximum dose, butfrom sample to sample there have been occasional shifts in the baselineintensities of the response at zero concentration.

For these experiments, either 10 or 100 TNFα-treated 3T3 or SNB19cells/well were tested. On the basis of the standard deviations measuredfor these samples, and by taking t-values for the student's t-test, wehave estimated the minimum detectable doses for each case of cell type,compound, number of cells per well, and for different choices of howmany wells are sampled per condition. The latter factor determines thenumber of degrees of freedom that are provided in the sample of data.Increasing the number of wells from 4 to 16, and increasing the numberof cells per well from 10 to 100, improves the minimum detectable dosesconsiderably. For 3T3 cells, which show lower minimum detectable dosesthan the SNB19 cells, and for the case of 1% false negative and 1% falsepositive rates, we estimate that 100 cells per well and a sampling of 12or 16 wells are sufficient to detect a dose approximately equal to theEC50 value of 0.15 ng/ml. If the false positive rate is relaxed to 20%,a concentration of approximately half that value can be detected (0.83ng/ml). One hundred cells can conveniently be sampled over a cellculture surface area of less than 1 mm². TABLE 3 EC50 levels for TNFαand IL-1α (based on molecular weights of 17 kD for both) Compound CellType EC50 (10⁻¹² moles/liter) TNFα 3T3 8.8 SNB19 5.9 IL-1α 3T3 0.24SNB19 59

3. Phospho-p38 Mitogen Activated Protein Kinase (pp38MAPK)

MAPKs play a role in not only cell growth and division, but as mediatorsof cellular stress responses. One MAPK, p38, is activated by chemicalstress inducers such as hyper-osmolar sorbitol, hydrogen peroxide,arsenite, cadmium ions, anisomycin, sodium salicylate, and LPS.Activation of p38 is also accompanied by its translocation into thenucleus from the cytoplasm.

MAPK p38 lies in a pathway that is a cascade of kinases. Thus, p38 is asubstrate of one or more kinases, and it acts to phosphorylate one ormore substrates in time and space within the living cell.

The assay we present here measures, as one of its parameters, p38activation using immunolocalization of the phosphorylated form of p38 intoxin-treated cells. The assay was developed to be flexible enough toinclude the simultaneous measurement of other parameters within the sameindividual cells. Because the signal transduction pathway mediated bythe transcription factor NF-κB is also known to be involved in the cellstress response, we included the activation of NF-κB as a secondparameter in the same assay.

-   -   Our experiments demonstrate an immunofluorescence approach can        be used to measure p38 MAPK activation either alone or in        combination with NF-κB activation in the same cells. Multiple        cell types, model toxins, and antibodies were tested, and        significant stimulation of both pathways was measured in a        high-content mode. The phospho-p38 antibodies used in this study        were purchased from Sigma Chemical Company (St. Louis, Mo.). We        report that at least two cell stress signaling pathways can not        only be measured simultaneously, but are differentially        responsive to classes of model toxins. FIG. 46 shows the        differential response of the p38 MAPK and NF-κB pathways across        three model toxins and two different cell types. Note that when        added alone, three of the model toxins (IL1α, TNFα and        Anisomycin) can be differentiated by the two assays as        activators of specific pathways.

IκB Chimera

-   -   IkB degradation is the key event leading to nuclear        translocation of NF-kB and activation of the NFkB-mediated        stress response. We have chosen this sensor to complement the        NF-kB sensor as a classifier in a high-throughput mode: the        measurement of loss of signal due to degradation of the IκB-GFP        fusion protein requires no spatial resolution within individual        cells, and as such we envision IkB degradation measurements        being made rapidly on an entire cell substrate.

This biosensor is based on fusion of the first 60 amino acids of IkB tothe Fred25 variant of GFP. This region of IkB contains all theregulatory sequences, including phosphorylation sites and ubiquitinationsites, necessary to confer proteosome degradation upon the biosensor.Knowing this, stimulation of any pathway that would typically lead toNFkB translocation results in degradation of this biosensor. Monitoringthe fluorescence intensity of cells expressing IkB-GFP identifies thedegradation process.

Examples of Identifiers:

In our toxin identification strategy, the first two levels ofcharacterization ensure a rapid readout of toxin class withoutsacrificing the ability to detect many new mutant toxins or dissectseveral complex mixtures of known toxins. The third level of biosensorsare identifiers, which can identify a specific toxin or group of toxins.In one embodiment, an identifier comprises a protease biosensor thatresponds to the activity of a specific toxin. Other identifiers areproduced with reporters/biosensors specific to their activities. Theseinclude, but are not limited to post-translational modifications such asphosphorylation or ADP-ribosylation, translocation between cellularorganelles or compartments, effects on specific organelles or cellularcomponents (for example, membrane permeabilization, cytoskeletonrearrangement, etc.)

ADP-ribosylating toxins—These toxins include Pseudomonas toxin A,diphtheria toxin, botulinum toxin, pertussis toxin, and cholera toxin.For example, C. botulinum C2 toxin induces the ADP-ribosylation ofArg177 in the cytoskeletal protein actin, thus altering its assemblyproperties. Besides the construction of a classifier assay to measureactin-cytoskeleton regulation, an identifier assay can be constructed todetect the specific actin ADP-ribosylation. Because the ADP-ribosylationinduces a conformational change that no longer permits the modifiedactin to polymerize, this conformational change can be detectedintracellularly in several possible ways using luminescent reagents. Forexample, actin can be luminescently labeled using a fluorescent reagentwith an appropriate excited state lifetime that allows for themeasurement of the rotational diffusion of the intracellular actin usingsteady state fluorescence anisotropy. That is, toxin-modified actin willno longer be able to assemble into rigid filaments and will thereforeproduce only luminescent signals with relatively low anisotropy, whichcan be readily measured with an imaging system. In another embodiment,actin can be labeled with a polarity-sensitive fluorescent reagent thatreports changes in actin-conformation through spectral shifts of theattached reagent. That is, toxin-treatment will induce a conformationalchange in intracellular actin such that a ratio of two fluorescencewavelengths will provide a measure of actin ADP-ribosylation.

Cytotoxic phospholipases—Several gram-positive bacterial species producecytotoxic phospholipases. For example, Clostridium perfringens producesa phospholipase C specific for the cleavage of phosphoinositides. Thesephosphoinositides (e.g., inositol 1,4,5-trisphosphate) induce therelease of calcium ions from intracellular organelles. An assay that canbe conducted as either high-content or high-throughput can beconstructed to measure the release of calcium ions using fluorescentreagents that have altered spectral properties when complexed with themetal ion. Therefore, a direct consequence of the action of aphospholipase C based toxin can be measured as a change in cellularcalcium ion concentration.

Exfoliative toxins—These toxins are produced by several Staphylococcalspecies and can consist of several serotypes. A specific identifier forthese toxins can be constructed by measuring the morphological changesin their target organelle, the desmosome, which occur at the junctionsbetween cells. The exfoliative toxins are known to change the morphologyof the desmosomes into two smaller components called hemidesmosomes. Inthe high-content assay for exfoliative toxins, epithelial cells whosedesmosomes are luminescently labeled are subjected to image analysis. Anmethod that detects the morphological change between desmosomes andhemidesmosomes is used to quantify the activity of the toxins on thecells.

Most of these identifiers can be used in high throughput assaysrequiring no spatial resolution, as well as in high content assays.

Several biological threat agents act as specific proteases, and thus wehave focused on the development of fluorescent protein biosensors thatreport the proteolytic cleavage of specific amino acid sequences foundwithin the target proteins.

A number of such protease biosensors (including FRET biosensors) aredisclosed above, such as the caspase biosensors, anthrax, tetanus,Botulinum, and the zinc metalloproteases. FRET is a powerful techniquein that small changes in protein conformation, many of which areassociated with toxin activity, can not only be measured with highprecision in time and space within living cells, but can be measured ina high-throughput mode, as discussed above.

As described above, one of skill in the art will recognize that theprotease biosensors of this aspect of the invention can be adapted toreport the activity of any protease, by a substitution of theappropriate protease recognition site. These biosensors can be used inhigh-content or high throughput screens to detect in vivo activation ofenzymatic activity by toxins, and to identify specific activity based oncleavage of a known recognition motif. These biosensors can be used inboth live cell and fixed end-point assays, and can be combined withadditional measurements to provide a multi-parameter assay.

Anthrax LF

Anthrax is a well-known agent of biological warfare and is an excellenttarget for development of a biosensor in the identifier class. Lethalfactor (LF) is one of the protein components that confer toxicity toanthrax, and recently two of its targets within cells were identified.LF is a metalloprotease that specifically cleaves Mek1 and Mek2proteins, kinases that are part of the MAP-kinase signaling pathway.Green fluorescent protein (GFP) is fused in-frame at the amino terminusof either Mek1 or Mek2 (or both), resulting in a chimeric protein thatis retained in the cytoplasm due to the presence of a nuclear exportsequence (NES) present in both of the target molecules. Upon cleavage byactive lethal factor, GFP is released from the chimera and is free todiffuse into the nucleus. Therefore, measuring the accumulation of GFPin the nucleus provides a direct measure of LF activity on its naturaltarget, the living cell.

Multi-Parametric Screens

Multiparameter high-content assays for toxin activity within a singlepopulation of cells has several advantages over single parameter assays:

-   1. The reagent and cell-handling overhead is greatly reduced over    multiple single parameter assays.-   2. The time requirements for sample preparation and analysis are    greatly reduced.-   3. Reagents can be conserved.-   4. Most importantly, the biological analysis and the building of    cellular knowledge can be greatly enhanced when multiple toxin    activity parameters are measured within single cells.

Presented here is an analysis of multiple physiological targets withinmultiple single cell types. The results are consistent with toxinidentification and characterization. The specific toxins chosen for thedemonstration were Staphylococcus Enterotoxin B, (SEB), tumor necrosisfactor alpha (TNFα), and botulinum toxin.

To complete the analysis, the following strategy was pursued:

-   1. Choose intracellular targets known to be modulated by SEB and    botulinum toxins and substitute analogs that can be used to verify    the assay. SEB is known to activate p38 MAPK in some cell types. The    botulinum toxin C3 exoenzyme is known to affect the organization of    the actin-cytoskeleton through its ADP-ribosylation activity. One    well-characterized effect of TNFα is the stimulation of NF-κB    transcriptional activity. Furthermore, it is also known that there    is cross talk between the pathways activated by all three of the    above toxins. Therefore, a combinatorial approach was used to    measure the activation of cytoskeletal, MAPK, and NF-κB pathways    within the same single cells.-   2. Choose toxin analogs known to stimulate the above pathways. To    simulate the activity of the above toxins, the following compounds    and ions were proposed for use as toxin analogs:    -   i) Anisomycin [SEB analog]—This drug is known to activate the        p38 MAPK pathway through increased phosphorylation of p38.        Measurement is done using antibodies against the phosphorylated        (activated) form of p38 and the cytoplasm to nuclear        translocation application.    -   ii) TNFα [TNFα]—This cytokine is known to activate the NF-κB        pathway and is measurable as the cytoplasm to nuclear        translocation of the transcription factor.    -   iii) Latrunculin [botulinum C3 exoenzyme analog]—This drug is        known to affect the equilibrium between globular and filamentous        actin in living cells. This shift in equilibrium is measurable        as a change in the intensity of fluorescent phalloidin labeling        of single cells.

Cell plating and drug treatment. Swiss 3T3 cells were plated at 15,000cells per well in a 96-well microplate 8 hours before drug treatment.Columns consisting of 8 wells within the plate were treated with thefollowing concentration ranges of drugs: TNFα, 0.781-10 ng/ml;anisomycin, 7.81-1000 nM; and latrunculin, 2.5-320 nM. The cells weretreated with the drugs for 30 minutes at 37° C. in a humidified 5% CO₂atmosphere.

Cell labeling. After drug incubation, the medium was removed from thewells and replaced with a solution (175 μl/well) containing 4%formaldehyde and 10 μg/ml Hoechst 33342 in Hank's Balanced Salt Solution(HBSS). The cells were incubated in this solution for 20 minutes at roomtemperature. The wells were rinsed once with HBSS (200 μl/well), andthen treated for 5 minutes at room temperature with a solution (200μl/well) containing 0.5% (v/v) Triton X-100 in HBSS. After thisdetergent extraction, the cells were rinsed as above and incubated witha solution (50 μl/well) containing a 1:500 dilution of a mousemonoclonal antibody to phosphorylated p38 MAPK, a 1:200 dilution of arabbit polyclonal antibody to NF-κB, and a 1:400 dilution of Alexa 488labeled phalloidin for 1 hour at room temperature. After rinsing asabove, the cells were incubated one last time with a solution (50μl/well) containing a 1:150 dilution of Alexa 568-labeled polyclonalanti-mouse antibody solution and a 1:150 dilution of a Cy5-labeledpolyclonal anti-rabbit antibody solution for 1 hour at room temperature.The cells were rinsed as above, and stored at 4° C. in a sealed platecontaining HBSS (200 μl/well).

High-content analysis of labeled cells. The 96-well microplatecontaining the multiply labeled cells was scanned with a cell screeningsystem, including but not limited to those disclosed in U.S. Pat. No.5,989,835 and in U.S. patent application Ser. No. 09/031,271 filed Feb.27, 1998, both incorporated by reference herein in their entirety.Cytoplasm to nuclear ratios were made for p38 MAPK, NF-κB, and actinwithin the same population of cells.

Results and Discussion

Table 4 shows the results of the multiparameter measurement of threeintracellular activities as a function of the concentration of threedifferent toxins. NF-κB translocation is shown to be sensitive to TNFαconcentration, but p38 MAPK and actin-cytoskeleton assembly state in thesame cells are relatively insensitive. p38 MAPK activation is moresensitive than NF-κB translocation to increasing anisomycinconcentration. Here again, actin-cytoskeletal assembly state isrelatively unaffected by this drug. High concentrations of latrunculininduce a reorganization of the actin-cytoskeleton, but have relativelylittle effect on p38 MAPK activation or NF-κB translocation in the samecells. TABLE 4 Maximal Cellular Response Relative to Control* AnalogNF-κB p38 MAPK Actin TNFα 1.8 (>25 nM) 1.2 (>200 nM) 1.0 Anisomycin 1.01.4 (>200 nM) 1.0 Latrunculin 1.0 1.0 1.2 (>150 nM)*A maximal cellular response of 1.0 indicates no significant differenceover control values.

The analysis of cellular data using multiparameter high-content assaysdemonstrated here provides new insights into the mechanisms of bothknown and unknown toxin activity. For example, TNFα acts as a toxinthrough a molecular pathway that uses NF-κB translocation to activatespecific cellular genes, while anisomycin action is consistent with theactivation of both p38 MAPK and NF-κB pathways. The further applicationof this multiparameter approach where comparisons are made at the singlecell level also greatly increases the resolution to which toxin actioncan be dissected and therefore improves the precision and sensitivity oftoxin detection.

EXAMPLE 12 Matrix for Toxin Detection and Organ Localization

In another aspect, the present invention provides a cassette for cellscreening, comprising a substrate having a surface; and a fluid deliverysystem mated with the substrate, wherein the fluid delivery systemcomprises (1) a matrix of openings or depressions, wherein each regionof the substrate enclosed by the opening or depression in the matrixcomprises an individually addressable domain; and (2) microfluidicchannels that supply fluid to the domains. The architecture of thefluidic channels is governed by the laws of fluid flow in microchannelsalone.

In this aspect, the microfluidic device consists of a matrix of eitheropenings or depressions with dimensions ranging from 1 μm to 10,000 μmper side, or 1 μm to 10,000 μm in diameter with depths ranging from 10μm to 10,000 μm. Such a matrix can comprise any desired number of rowsand columns.

In one embodiment, a 3×3 matrix comprises openings, which when bonded tothe substrate permits modification of the substrate surface withadhesion chemistry and cells. Subsequently, the openings are closed, andthe closed cassette is used for screening by pumping fluids and reagentsinto the assay domains via the microfluidic channels. The microfluidicdevice can be bonded to the substrate via such non-limiting means asanodic bonding, electrofusion, thermal bonding, adhesive bonding, andpressure bonding. Each region on the substrate enclosed by an opening ordepression now constitutes a “domain” on the substrate.

In another embodiment of this aspect of the invention, the substratecomprises cell binding locations separated by cell repulsive regions,and cells are arrayed on the cell binding locations. In a preferredembodiment, each domain on the substrate comprises a single cell type.The nine domains on the substrate are thus preferably arrayed withbetween one and nine different cell types, with each cell type bearingone or more biosensors reporting on one or more intracellular pathways.In further preferred embodiments, at least three different cell typesare arrayed on the substrate; and the different cell types arrayed onthe substrate are specific for different tissue types, including but notlimited to cells specific for connective tissue, neuronal tissue, andthe immune system.

As such, each of the nine domains has a single cell type arrayed on cellbinding locations. Each of the nine domains are individually addressableby a fluid of choice using the microfluidic device. If the fluiddelivery device comprises a matrix of openings, the top of the device issealed from the environment with a glass or plastic film, or an airpermeable and water impermeable membrane such as BREATHEASY™ (SigmaChemical Co.), by means such as, but not limited to anodic bonding,anodic bonding, electrofusion, thermal bonding, adhesive bonding, andpressure bonding. The semi-permeable membrane enables oxygen and carbondioxide diffusion for long-term viability and functionality of thecells.

Cell adhesion promoters and inhibitors that can be used include, but arenot limited to, those discussed throughout the application. In a mostpreferred embodiment, the cell adhesion promoter comprises a silane, andthe cell repulsive moiety comprises tresyl-activated PEG. The surfacemodifications can be achieved by either transferring cell adhesivemolecules onto the substrate in select regions, or by coating selectregions of the substrate with cell repulsive molecules via any method,including but not limited to those described above. The substrate somodified is arrayed with cells in each of the regions enclosed by thesquare openings or depressions. Alternatively, the substrate can bemodified with cell adhesive and cell selective chemistries after bondingto the microfluidic device. Where the fluid delivery system comprises amatrix of openings, the chemistries and cells can be arrayed on thesubstrate either via the microfluidic channels, or via the openings inthe fluid delivery system, after which the wells are closed with a glassfilm, plastic film, or air permeable and water impermeable membrane asdescribed above. Where the fluid delivery system comprises a matrix ofdepressions, the chemistries and cells can be delivered via themicrofluidic channels.

In another aspect, the invention provides a method for cell screening,comprising providing the cassette for cell screening described in thisexample, providing an optical system to obtain images of the cells;contacting the domains with a test compound; and obtaining images of thecells to determine an effect of the test compound on the cells. Suchscreening can be conducted in either a high content mode, a highthroughput mode or a combination of the two. The high throughput modepermits optical detection of signals arising from all domains on thesubstrate in one pass, while the high content mode permits sub-cellularresolution and detection of signals arising from each individual cellwithin each domain.

In a preferred embodiment, the method detects the pathway and organlocalization of a toxin, wherein the substrate comprises multiple celltypes each with one or more biosensors that are affected by a toxin;contacting the domains with a test sample potentially comprising atoxin, and obtaining images of the cells to determine an effect of thetoxin on the different cell types, wherein an effect of the toxin on thecell types indicates the toxin pathway and organ localization. As usedherein, “organ localization” means that a particular toxin has an effecton cells from a particular organ, and thus its toxicity may be mediated,at least in part, through that organ. The term does not imply that cellsfrom other organs are not affected by the toxin.

In a further preferred embodiment, the biosensors expressed by the cellsare selected from the group consisting of detectors, classifiers, andidentifiers, as described above.

In a further aspect, the invention further provides an automated methodfor cell based toxin detection and organ localization comprising

providing an array of locations containing cells to be treated with atest substance, wherein the array comprises at least a first cell typeand a second cell type, and wherein the first cell type and the secondcell type are not contained on the same location in the array; whereinthe first and second cell types are derived from different organ types;wherein each of the cell types comprises at least one luminescentreporter molecule; wherein the localization, distribution, structure, oractivity of the at least one luminescent reporter molecule is altered bya toxin to be detected;

contacting the at least first cell type and second cell type with thetest substance either before or after possession of the at least oneluminescent reporter molecules by the first cell type and the secondcell type;

imaging or scanning multiple cells in each of the locations containingthe first cell type or the second cell type to obtain luminescentsignals from the luminescent reporter molecule in the first cell typeand the second cell type;

converting the luminescent signals into digital data;

utilizing the digital data to automatically measure the localization,distribution, structure or activity of the at least one luminescentreporter molecule on or in the first cell type and the second cell type,wherein a change in the localization, distribution, structure oractivity of the luminescent reporter molecule indicates the presence ofa toxin and an organ localization of the toxin.

In a preferred embodiment, the array further comprise a third cell type,wherein the first, second, and third cell types are not contained on thesame location in the array; and wherein the first, second, and thirdcell types are derived from different organ types.

In a further preferred embodiment, the cell types further comprise atleast a second luminescent reporter molecule; wherein the localization,distribution, structure, or activity of the second luminescent reportermolecule is altered by a toxin to be detected.

For developing a 3×3 matrix of assays and toxin analogs formulti-parametric toxin identification and organ localization, severalfactors were considered, including the cellular events or pathways thatare affected by toxic agents, the critical biochemical targets thatundergo a measurable change in response to a toxin, and whether suchchanges in the critical biochemical target are measurable. The followingare representative examples of such assays, toxins, and toxin analogsthat can be used in accordance with the present invention. One of skillin the art will recognize that many other such assays, toxins, and toxinanalogs can be used according to the teachings of the present invention,all of which are encompassed by the-present invention;

The shape of a cell is determined in large part by its actincytoskeleton. Upon loss of F-actin, cells undergo profound shapechanges. Botulinum C3 toxin is a member of a class of toxins that targetthe Ras signaling pathway. Botulinum C3 toxin inactivates Rho, leadingto actin depolymerization, thus causing a detectable change in cellmorphology, particularly a decrease in cell area. Similarly to BotulinumC3, cytochalasin D binds to actin and prevents polymerization, leadingto loss of cytoskeletal organization. Thus, cytochalasin D can be usedas a Botulinum C3 analog to demonstrate the ability of the toxin assayto classify a toxin present in the sample as affecting cell morphologyin a manner similar to Botulinum C3. F-actin can be labeled withfluorescent phalloidin to reveal the cytoskeletal structure, and changesin such structure can be measured in response to a test samplecontaining cytochalasin D. Thus, the present assay serves to classifythe toxin as one that perturbs the cell's actin filament structure.

Botulinum C3 also induces the expression of cell stress proteins, andthus would be detected as a toxin by a cell stress protein biosensor,such as those described above. Botulinum C3 also affects membranetrafficking.

The p38 MAP kinase, (pp38MAPK, discussed above), is phosphorylated andtranslocates to the nucleus upon activation of the MAP kinase pathway.Antibodies specific for phosphorylated p38 (the target of pp38MAPK)report its location in the cell. pp38MAPK is also a target ofStaphylococcal enterotoxin B (SEB) and T2 Toxin. SEB alters the functionof immune cells and stimulates p38 activation and translocation in someT-cell lines. Certain tricothecene mycotoxins (e.g. T2 toxins such as T2triol) inhibit protein synthesis and induce p38 activity. Anisomycinalso inhibits protein synthesis and induces p38 translocation to thenucleus as discussed above, and thus can be used as an analog of SEB andT2 toxin, to demonstrate the ability of the toxin assay to classify atoxin present in the sample as affecting p38 activation and/orlocalization in a manner similar to SEB and/or T2 toxins. Furthermore,by utilizing cells specific for the immune system, the assay providesinformation on toxin localization in the body.

SEB also induces the expression of cell stress proteins, and thus couldbe detected in a test sample as a toxin by a cell stress proteinbiosensor, such as those described above. SEB also affects microtubulestructure, and its presence in a test sample would lead to aclassification of the test sample as containing a toxin that perturbsthe cell microtubule structure.

Cells respond to environmental agents of stress (as discussed above)through specific signaling cascades. As discussed above, NFkB is atranscription factor that activates production of proteins as part ofthe cellular defense response to general cell stress. NFkB resides inthe cytoplasm but translocates to the nucleus upon release from acomplex with IκB. Antibody staining reveals the subcellular localizationof NFkB. TNF-α is a strong inducer of NF-κB translocation to thenucleus, and thus can be used as an analog of toxins that induce cellstress, including but not limited to cholera toxin. TABLE 5 Matrix 1:Dissection of pathway and organ localization of 1 toxin per cassetteToxin 1 Toxin 1 Toxin 1 1 2 3 A SD3T3 (HSP) SD3T3 (NF-kB) SD3T3(β-tubulin) B SNB19 (HSP) SNB19 (NF-kB) SNB19 (β-tubulin) C RAW (HSP)RAW (NF-kB) RAW (β-tubulin)

This example permits addressing the effect of a single toxin onconnective tissue, neuronal tissue, and immune specific cells asmeasured through cell stress, transcription factor activation, andcytoskeleton reorganization. Each cassette has a 3×3 matrix of squareopenings, and a total of nine domains. Three different cell types areeach arrayed in three of the domains, with the cells in a single domainexpressing a single toxin biosensor. Each cell type expresses adifferent toxin biosensor in each of the domains in which it is arrayed.

Cell Types:

(A) SD3T3: connective tissue cell type: Serum-deprived 3T3 fibroblast(ATCC CCL-92)

(B) SNB19: neuronal tissue cell type: Human glioblastoma (ATCC#CRL-2219)

(C) RAW: Immune specific cell type: Murine macrophage cell line(ATCC#TIB-71)

Column 1 (A1/B1/C1) will provide a “low resolution” image of the sample.If the sample contains the minimum detectable concentration of eitherSEB, Cholera Tox, or Bot. Tox, it will elicit a positive cell stressresponse from each of the 3 tissue specific cells. A positive responsein Column 1 would entail a “higher resolution” probe of the sample inColumns 2 and 3. The modality of action and tissue specificity of thetoxin are dissected in Columns 2 and 3, using the NF-kB and β-tubulinbiosensors. TABLE 6 Matrix 2: Dissection of pathway and organlocalization of 1 toxin per cassette plus an internal control Toxin 1Toxin 1 PBS 1 2 3 A SD3T3 SD3T3 (NF-kB + SD3T3 (NF-kB + (HSP) β-tubulin)β-tubulin) B SNB19 SNB19 (NF-kB + SNB19 (NF-kB + (HSP) β-tubulin)β-tubulin) C RAW RAW (NF-kB + β-tubulin) RAW (NF-kB + β-tubulin) (HSP)

Each cassette with a 3×3 array of the 3 cell types with each of the 3toxin biosensors enables addressing the effect of a single toxin onconnective tissue, neuronal tissue, and immune specific cells asmeasured through cell stress, transcription factor activation, andcytoskeleton reorganization. Columns 2 and 3 have cells expressing 2fluorescent protein biosensors each. This enables simultaneous detectionof 2 different sets of macromolecules representing two differentpathways within the same cells. TABLE 7 Matrix 3: Dissection of pathwayand organ localization of 3 toxins per cassette Toxin 1 Toxin 2 Toxin 31 2 3 A SD3T3 (HSP + NF- SD3T3 (HSP + NF-kB + β- SD3T3 (HSP + NF-kB + β-kB + β-tubulin) tubulin) tubulin) B SNB19 (HSP + NF- SNB19 (HSP +NF-kB + β- SNB19 (HSP + NF-kB + β- kB + β-tubulin) tubulin) tubulin) CRAW (HSP + NF- RAW(HSP + NF-kB + β- RAW (HSP + NF-kB + β- kB +β-tubulin) tubulin) tubulin)

Each cell on the substrate has the capacity to simultaneously report theeffect of a toxin on cell stress, transcription factor activation andtranslocation, and reorganization of the cytoskeletal proteins.

Experimental Design and Results of 3×3 Matrix Model System

The following matrix of toxins, analogs, and assays were used for 3×3matrix study based on the above examples, except that the Swiss 3T3cells were exposed to the toxin analogs for 45 minutes. In each assay,the analogs were used at the following concentrations: Medium exchangenegative control TNF α 1.8 nM Anisomycin  30 μM Cytochalasin D   2 μM

Cell Stress Target

-   -   Quantitative results were determined based on data obtained from        1500 cells totaled across 16 wells. The proportion of NF-kB        protein in the nucleus versus the cytoplasm for wells treated by        the toxin analogs is shown in FIG. 47. The dominant response was        obtained for TNF α.

SEB/T2 Target

-   -   Quantitative results were determined based on data obtained from        1500 cells totaled across 16 wells. Phosphorylated p38 MAPK in        the nucleus versus the cytoplasm for wells treated by the toxin        analogs was as shown in FIG. 48. The dominant response was        obtained for anisomycin.

Botulinum C2 Target

Quantitative results were obtained based on results from 500 cellstotaled across 6 wells. Cell area for cells treated with the toxinanalogs was as shown in FIG. 49. The dominant response was obtained forcytochalasin D.

The present invention is not limited by the aforementioned particularpreferred embodiments. It will occur to those ordinarily skilled in theart that various modifications may be made to the disclosed preferredembodiments without diverting from the concept of the invention. Allsuch modifications are intended to be within the scope of the presentinvention.

1. A machine readable storage medium comprising a program containing a set of instructions for causing a cell screening system to execute procedures for detecting a cell based toxin and organ localization comprising: a) imaging or scanning an array of locations containing cells contacted with a test substance, to obtain luminescent signals from a luminescent reporter molecule in the cells; wherein the array comprises at least a first cell type and a second cell type, and wherein the first cell type and the second cell type are not contained on the same location in the array; wherein the first and second cell types are derived from different organ types; wherein each of the cell types comprises at least a first luminescent reporter molecule; wherein a localization, distribution, structure, and/or activity of the at least first luminescent reporter molecule is altered by a toxin to be detected; and wherein the contacting occurs either before or after possession of the at least one luminescent reporter molecules by the first cell type and the second cell type; b) converting the luminescent signals into digital data; c) utilizing the digital data to automatically measure the localization, distribution, structure and/or activity of the at least one luminescent reporter molecule on or in the first cell type and the second cell type, wherein a change in the localization, distribution, structure or activity of the luminescent reporter molecule indicates the presence of a toxin and an organ localization of the toxin.
 2. The machine readable storage medium of claim 1, wherein the array further comprises a third cell type, wherein the first, second, and third cell types are not contained on the same location in the array; and wherein the first, second, and third cell types are derived from different organ types wherein each of the cell types comprises at least a first luminescent reporter molecule; wherein a localization, distribution, structure, and/or activity of the at least first luminescent reporter molecule is altered by a toxin to be detected.
 3. The machine readable storage medium of claim 1 wherein the one or more of the cell types further comprises at least a second luminescent reporter molecule; wherein the localization, distribution, structure, and/or activity of the second luminescent reporter molecule is altered by a toxin to be detected.
 4. The machine readable storage medium of claim 3 wherein one or more of the cell types further comprises at least a third luminescent reporter molecule; wherein the localization, distribution, structure, or activity of the third luminescent reporter molecule is altered by a toxin to be detected.
 5. The machine readable storage medium of claim 3, wherein the at least first luminescent reporter molecule is a detector, wherein the detector detects a toxin present in the test substance; and the at least second luminescent reporter molecule is selected from the group consisting of a classifier or an identifier, wherein the classifier detects a toxin present in the test substance and identifies a cell pathway affected by a toxin present in the test substance, and the identifier detects the presence of a toxin present in the test substance and identifies a specific toxin or group of toxins present in the test substance.
 6. The machine readable storage medium of claim 4, wherein the at least first luminescent reporter molecule is a detector, wherein the detector detects a toxin present in the test substance; the at least second luminescent reporter molecule is a classifier, wherein the classifier detects a toxin present in the test substance and identifies a cell pathway affected by a toxin present in the test substance; and the at least third luminescent reporter molecule is an identifier, wherein the identifier detects the presence of a toxin present in the test substance and identifies a specific toxin or group of toxins present in the test substance.
 7. The machine readable storage medium of claim 5, wherein the second luminescent reporter is a classifier, and the digital data derived from the classifier is used to select an identifier for identification of the specific toxin or group of toxins.
 8. The machine readable storage medium of claim 1, wherein the at least first luminescent reporter molecule is a fluorescent reporter molecule. 